SRX11386439 GSM5434068 SRP327554 SRS9431086 GSM5434068 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434068 GSM5434068 GEO Accession GSM5434068 SRX11386440 GSM5434069 SRP327554 SRS9431087 GSM5434069 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434069 GSM5434069 GEO Accession GSM5434069 SRX11386441 GSM5434070 SRP327554 SRS9431088 GSM5434070 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434070 GSM5434070 GEO Accession GSM5434070 SRX11386442 GSM5434071 SRP327554 SRS9431090 GSM5434071 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434071 GSM5434071 GEO Accession GSM5434071 SRX11386443 GSM5434072 SRP327554 SRS9431089 GSM5434072 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434072 GSM5434072 GEO Accession GSM5434072 SRX11386444 GSM5434073 SRP327554 SRS9431091 GSM5434073 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434073 GSM5434073 GEO Accession GSM5434073 SRX11386445 GSM5434074 SRP327554 SRS9431092 GSM5434074 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434074 GSM5434074 GEO Accession GSM5434074 SRX11386446 GSM5434075 SRP327554 SRS9431093 GSM5434075 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434075 GSM5434075 GEO Accession GSM5434075 SRX11386447 GSM5434084 SRP327554 SRS9431094 GSM5434084 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434084 GSM5434084 GEO Accession GSM5434084 SRX11386448 GSM5434085 SRP327554 SRS9431095 GSM5434085 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434085 GSM5434085 GEO Accession GSM5434085 SRX11386449 GSM5434086 SRP327554 SRS9431096 GSM5434086 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434086 GSM5434086 GEO Accession GSM5434086 SRX11386450 GSM5434087 SRP327554 SRS9431097 GSM5434087 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434087 GSM5434087 GEO Accession GSM5434087 SRX11386451 GSM5434088 SRP327554 SRS9431098 GSM5434088 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434088 GSM5434088 GEO Accession GSM5434088 SRX11386452 GSM5434089 SRP327554 SRS9431099 GSM5434089 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434089 GSM5434089 GEO Accession GSM5434089 SRX11386453 GSM5434090 SRP327554 SRS9431100 GSM5434090 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434090 GSM5434090 GEO Accession GSM5434090 SRX11386454 GSM5434091 SRP327554 SRS9431102 GSM5434091 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434091 GSM5434091 GEO Accession GSM5434091 SRX11386455 GSM5434076 SRP327554 SRS9431101 GSM5434076 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434076 GSM5434076 GEO Accession GSM5434076 SRX11386456 GSM5434077 SRP327554 SRS9431103 GSM5434077 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434077 GSM5434077 GEO Accession GSM5434077 SRX11386457 GSM5434078 SRP327554 SRS9431104 GSM5434078 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434078 GSM5434078 GEO Accession GSM5434078 SRX11386458 GSM5434079 SRP327554 SRS9431105 GSM5434079 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434079 GSM5434079 GEO Accession GSM5434079 SRX11386459 GSM5434080 SRP327554 SRS9431106 GSM5434080 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434080 GSM5434080 GEO Accession GSM5434080 SRX11386460 GSM5434081 SRP327554 SRS9431107 GSM5434081 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434081 GSM5434081 GEO Accession GSM5434081 SRX11386461 GSM5434082 SRP327554 SRS9431108 GSM5434082 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434082 GSM5434082 GEO Accession GSM5434082 SRX11386462 GSM5434083 SRP327554 SRS9431109 GSM5434083 RNA-Seq TRANSCRIPTOMIC cDNA he mice were sacrificed on day 13 and pleural fluid was gently aspirated using a 1-ml syringe through the diaphragm, and its volume was measured with a 1 mL pipette. The pleural fluids were centrifuged at 500 g for 10 min and cell pellet was further treated with 2 mL ACK Lysing Buffer (Thermo Fisher) to delete red blood cells for 10 min at 4 °C in 15-mL conical centrifuge tube. 10 mL cold PBS was added to stop the lysis, and the cells were centrifuged at 500 g for 5 min. The total RNA from MPE cell pellets were extracted by TRIzol Reagent. For r pleural tumors, tissues were cut into small pieces in PBS on ice and further processed in 1 x tumor digestion buffer (1 mg/mL Collagenase IV, 0.1 mg/mL Hyaluronidase, and 20 U/mL DNAse) at 37 °C for 1 h under rotation. The cell suspension was gathered by filtering through cell strainer. After centrifuge at 500 g for 5 min, the cell pellet was washed once by 5 mL PBS followed by centrifuge at 500 g for 5 min. The pellet was treated with ACK lysing buffer to remove red blood cells. The total RNA from pleural tumor cell pellets were extracted by TRIzol Reagent as well. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing Illumina NovaSeq 6000 gds 305434083 GSM5434083 GEO Accession GSM5434083