SRX19230566 GSM7015128_r1 SRP483237 GSE253061 SRS16635192 GSM7015128 GSM7015128 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230567 GSM7015129_r1 SRP483237 GSE253061 SRS16635193 GSM7015129 GSM7015129 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230568 GSM7015166_r1 SRP483237 GSE253061 SRS16635195 GSM7015166 GSM7015166 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230569 GSM7015167_r1 SRP483237 GSE253061 SRS16635194 GSM7015167 GSM7015167 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230570 GSM7015168_r1 SRP483237 GSE253061 SRS16635196 GSM7015168 GSM7015168 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230571 GSM7015169_r1 SRP483237 GSE253061 SRS16635197 GSM7015169 GSM7015169 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230572 GSM7015170_r1 SRP483237 GSE253061 SRS16635198 GSM7015170 GSM7015170 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230573 GSM7015171_r1 SRP483237 GSE253061 SRS16635199 GSM7015171 GSM7015171 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230574 GSM7015172_r1 SRP483237 GSE253061 SRS16635200 GSM7015172 GSM7015172 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230575 GSM7015238_r1 SRP483237 GSE253061 SRS16635201 GSM7015238 GSM7015238 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230576 GSM7015239_r1 SRP483237 GSE253061 SRS16635202 GSM7015239 GSM7015239 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230577 GSM7015240_r1 SRP483237 GSE253061 SRS16635203 GSM7015240 GSM7015240 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230578 GSM7015241_r1 SRP483237 GSE253061 SRS16635204 GSM7015241 GSM7015241 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230579 GSM7015242_r1 SRP483237 GSE253061 SRS16635205 GSM7015242 GSM7015242 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230580 GSM7015243_r1 SRP483237 GSE253061 SRS16635206 GSM7015243 GSM7015243 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230581 GSM7015244_r1 SRP483237 GSE253061 SRS16635207 GSM7015244 GSM7015244 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230582 GSM7015260_r1 SRP483237 GSE253061 SRS16635208 GSM7015260 GSM7015260 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230583 GSM7015261_r1 SRP483237 GSE253061 SRS16635209 GSM7015261 GSM7015261 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230584 GSM7015262_r1 SRP483237 GSE253061 SRS16635210 GSM7015262 GSM7015262 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230596 GSM7015263_r1 SRP483237 GSE253061 SRS16635222 GSM7015263 GSM7015263 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230597 GSM7015264_r1 SRP483237 GSE253061 SRS16635223 GSM7015264 GSM7015264 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230729 GSM7015202_r1 SRP483237 GSE253061 SRS16635355 GSM7015202 GSM7015202 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230730 GSM7015203_r1 SRP483237 GSE253061 SRS16635356 GSM7015203 GSM7015203 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230731 GSM7015204_r1 SRP483237 GSE253061 SRS16635357 GSM7015204 GSM7015204 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230732 GSM7015205_r1 SRP483237 GSE253061 SRS16635359 GSM7015205 GSM7015205 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230733 GSM7015206_r1 SRP483237 GSE253061 SRS16635358 GSM7015206 GSM7015206 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230734 GSM7015207_r1 SRP483237 GSE253061 SRS16635360 GSM7015207 GSM7015207 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230735 GSM7015208_r1 SRP483237 GSE253061 SRS16635361 GSM7015208 GSM7015208 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230736 GSM7015254_r1 SRP483237 GSE253061 SRS16635362 GSM7015254 GSM7015254 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230737 GSM7015255_r1 SRP483237 GSE253061 SRS16635364 GSM7015255 GSM7015255 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230738 GSM7015256_r1 SRP483237 GSE253061 SRS16635363 GSM7015256 GSM7015256 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230739 GSM7015257_r1 SRP483237 GSE253061 SRS16635365 GSM7015257 GSM7015257 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230740 GSM7015258_r1 SRP483237 GSE253061 SRS16635367 GSM7015258 GSM7015258 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230741 GSM7015259_r1 SRP483237 GSE253061 SRS16635366 GSM7015259 GSM7015259 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230765 GSM7015224_r1 SRP483237 GSE253061 SRS16635391 GSM7015224 GSM7015224 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230766 GSM7015225_r1 SRP483237 GSE253061 SRS16635392 GSM7015225 GSM7015225 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230767 GSM7015226_r1 SRP483237 GSE253061 SRS16635393 GSM7015226 GSM7015226 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230768 GSM7015227_r1 SRP483237 GSE253061 SRS16635395 GSM7015227 GSM7015227 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230769 GSM7015228_r1 SRP483237 GSE253061 SRS16635394 GSM7015228 GSM7015228 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230770 GSM7015229_r1 SRP483237 GSE253061 SRS16635396 GSM7015229 GSM7015229 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230771 GSM7015230_r1 SRP483237 GSE253061 SRS16635397 GSM7015230 GSM7015230 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230772 GSM7015253_r1 SRP483237 GSE253061 SRS16635398 GSM7015253 GSM7015253 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230799 GSM7014930_r1 SRP483237 GSE253061 SRS16635426 GSM7014930 GSM7014930 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230800 GSM7014931_r1 SRP483237 GSE253061 SRS16635425 GSM7014931 GSM7014931 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230801 GSM7014932_r1 SRP483237 GSE253061 SRS16635427 GSM7014932 GSM7014932 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230802 GSM7014933_r1 SRP483237 GSE253061 SRS16635428 GSM7014933 GSM7014933 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230803 GSM7014934_r1 SRP483237 GSE253061 SRS16635429 GSM7014934 GSM7014934 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230804 GSM7014935_r1 SRP483237 GSE253061 SRS16635430 GSM7014935 GSM7014935 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230805 GSM7014943_r1 SRP483237 GSE253061 SRS16635431 GSM7014943 GSM7014943 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230806 GSM7014944_r1 SRP483237 GSE253061 SRS16635432 GSM7014944 GSM7014944 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230807 GSM7014945_r1 SRP483237 GSE253061 SRS16635433 GSM7014945 GSM7014945 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230808 GSM7014946_r1 SRP483237 GSE253061 SRS16635435 GSM7014946 GSM7014946 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230809 GSM7014947_r1 SRP483237 GSE253061 SRS16635434 GSM7014947 GSM7014947 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230810 GSM7014948_r1 SRP483237 GSE253061 SRS16635436 GSM7014948 GSM7014948 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230811 GSM7014949_r1 SRP483237 GSE253061 SRS16635437 GSM7014949 GSM7014949 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230812 GSM7014957_r1 SRP483237 GSE253061 SRS16635438 GSM7014957 GSM7014957 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230813 GSM7014958_r1 SRP483237 GSE253061 SRS16635439 GSM7014958 GSM7014958 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230814 GSM7014959_r1 SRP483237 GSE253061 SRS16635440 GSM7014959 GSM7014959 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230815 GSM7014960_r1 SRP483237 GSE253061 SRS16635441 GSM7014960 GSM7014960 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230816 GSM7014961_r1 SRP483237 GSE253061 SRS16635442 GSM7014961 GSM7014961 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230817 GSM7014962_r1 SRP483237 GSE253061 SRS16635443 GSM7014962 GSM7014962 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230818 GSM7014963_r1 SRP483237 GSE253061 SRS16635444 GSM7014963 GSM7014963 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230819 GSM7014964_r1 SRP483237 GSE253061 SRS16635445 GSM7014964 GSM7014964 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230832 GSM7014919_r1 SRP483237 GSE253061 SRS16635458 GSM7014919 GSM7014919 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230833 GSM7014920_r1 SRP483237 GSE253061 SRS16635459 GSM7014920 GSM7014920 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230834 GSM7014936_r1 SRP483237 GSE253061 SRS16635460 GSM7014936 GSM7014936 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230835 GSM7014937_r1 SRP483237 GSE253061 SRS16635461 GSM7014937 GSM7014937 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230836 GSM7014938_r1 SRP483237 GSE253061 SRS16635462 GSM7014938 GSM7014938 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230837 GSM7014939_r1 SRP483237 GSE253061 SRS16635463 GSM7014939 GSM7014939 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230838 GSM7014940_r1 SRP483237 GSE253061 SRS16635464 GSM7014940 GSM7014940 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230839 GSM7014941_r1 SRP483237 GSE253061 SRS16635465 GSM7014941 GSM7014941 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230840 GSM7014942_r1 SRP483237 GSE253061 SRS16635467 GSM7014942 GSM7014942 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230841 GSM7014972_r1 SRP483237 GSE253061 SRS16635466 GSM7014972 GSM7014972 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230842 GSM7014973_r1 SRP483237 GSE253061 SRS16635468 GSM7014973 GSM7014973 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230843 GSM7014974_r1 SRP483237 GSE253061 SRS16635469 GSM7014974 GSM7014974 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230844 GSM7014975_r1 SRP483237 GSE253061 SRS16635470 GSM7014975 GSM7014975 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' NextSeq 550 SRX19230845 GSM7014976_r1 SRP483237 GSE253061 SRS16635471 GSM7014976 GSM7014976 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230846 GSM7014977_r1 SRP483237 GSE253061 SRS16635472 GSM7014977 GSM7014977 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230847 GSM7014978_r1 SRP483237 GSE253061 SRS16635473 GSM7014978 GSM7014978 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230877 GSM7015092_r1 SRP483237 GSE253061 SRS16635503 GSM7015092 GSM7015092 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230878 GSM7015093_r1 SRP483237 GSE253061 SRS16635504 GSM7015093 GSM7015093 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230884 GSM7015087_r1 SRP483237 GSE253061 SRS16635510 GSM7015087 GSM7015087 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230885 GSM7015088_r1 SRP483237 GSE253061 SRS16635511 GSM7015088 GSM7015088 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230886 GSM7015089_r1 SRP483237 GSE253061 SRS16635513 GSM7015089 GSM7015089 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230887 GSM7015090_r1 SRP483237 GSE253061 SRS16635512 GSM7015090 GSM7015090 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230888 GSM7015091_r1 SRP483237 GSE253061 SRS16635514 GSM7015091 GSM7015091 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230891 GSM7015080_r1 SRP483237 GSE253061 SRS16635517 GSM7015080 GSM7015080 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230892 GSM7015081_r1 SRP483237 GSE253061 SRS16635518 GSM7015081 GSM7015081 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230893 GSM7015082_r1 SRP483237 GSE253061 SRS16635519 GSM7015082 GSM7015082 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230894 GSM7015083_r1 SRP483237 GSE253061 SRS16635520 GSM7015083 GSM7015083 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230895 GSM7015084_r1 SRP483237 GSE253061 SRS16635521 GSM7015084 GSM7015084 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230896 GSM7015085_r1 SRP483237 GSE253061 SRS16635522 GSM7015085 GSM7015085 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230897 GSM7015086_r1 SRP483237 GSE253061 SRS16635523 GSM7015086 GSM7015086 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230898 GSM7015094_r1 SRP483237 GSE253061 SRS16635524 GSM7015094 GSM7015094 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230899 GSM7015095_r1 SRP483237 GSE253061 SRS16635525 GSM7015095 GSM7015095 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230900 GSM7015096_r1 SRP483237 GSE253061 SRS16635526 GSM7015096 GSM7015096 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230901 GSM7015097_r1 SRP483237 GSE253061 SRS16635527 GSM7015097 GSM7015097 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230902 GSM7015098_r1 SRP483237 GSE253061 SRS16635528 GSM7015098 GSM7015098 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230903 GSM7015099_r1 SRP483237 GSE253061 SRS16635529 GSM7015099 GSM7015099 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230904 GSM7015100_r1 SRP483237 GSE253061 SRS16635530 GSM7015100 GSM7015100 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230913 GSM7015137_r1 SRP483237 GSE253061 SRS16635539 GSM7015137 GSM7015137 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230914 GSM7015138_r1 SRP483237 GSE253061 SRS16635540 GSM7015138 GSM7015138 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230915 GSM7015139_r1 SRP483237 GSE253061 SRS16635541 GSM7015139 GSM7015139 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230916 GSM7015140_r1 SRP483237 GSE253061 SRS16635542 GSM7015140 GSM7015140 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230917 GSM7015141_r1 SRP483237 GSE253061 SRS16635544 GSM7015141 GSM7015141 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230918 GSM7015142_r1 SRP483237 GSE253061 SRS16635543 GSM7015142 GSM7015142 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230919 GSM7015143_r1 SRP483237 GSE253061 SRS16635545 GSM7015143 GSM7015143 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230920 GSM7015144_r1 SRP483237 GSE253061 SRS16635546 GSM7015144 GSM7015144 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230921 GSM7015152_r1 SRP483237 GSE253061 SRS16635547 GSM7015152 GSM7015152 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230922 GSM7015153_r1 SRP483237 GSE253061 SRS16635548 GSM7015153 GSM7015153 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230923 GSM7015154_r1 SRP483237 GSE253061 SRS16635549 GSM7015154 GSM7015154 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230924 GSM7015182_r1 SRP483237 GSE253061 SRS16635550 GSM7015182 GSM7015182 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230925 GSM7015183_r1 SRP483237 GSE253061 SRS16635551 GSM7015183 GSM7015183 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230926 GSM7015184_r1 SRP483237 GSE253061 SRS16635552 GSM7015184 GSM7015184 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230927 GSM7015185_r1 SRP483237 GSE253061 SRS16635553 GSM7015185 GSM7015185 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230928 GSM7015186_r1 SRP483237 GSE253061 SRS16635554 GSM7015186 GSM7015186 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230929 GSM7015187_r1 SRP483237 GSE253061 SRS16635555 GSM7015187 GSM7015187 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230949 GSM7015155_r1 SRP483237 GSE253061 SRS16635575 GSM7015155 GSM7015155 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230950 GSM7015156_r1 SRP483237 GSE253061 SRS16635576 GSM7015156 GSM7015156 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230951 GSM7015157_r1 SRP483237 GSE253061 SRS16635577 GSM7015157 GSM7015157 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230952 GSM7015158_r1 SRP483237 GSE253061 SRS16635578 GSM7015158 GSM7015158 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19230953 GSM7015181_r1 SRP483237 GSE253061 SRS16635579 GSM7015181 GSM7015181 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231075 GSM7015188_r1 SRP483237 GSE253061 SRS16635701 GSM7015188 GSM7015188 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231076 GSM7015189_r1 SRP483237 GSE253061 SRS16635702 GSM7015189 GSM7015189 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231077 GSM7015190_r1 SRP483237 GSE253061 SRS16635703 GSM7015190 GSM7015190 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231078 GSM7015191_r1 SRP483237 GSE253061 SRS16635704 GSM7015191 GSM7015191 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231079 GSM7015192_r1 SRP483237 GSE253061 SRS16635705 GSM7015192 GSM7015192 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231080 GSM7015193_r1 SRP483237 GSE253061 SRS16635706 GSM7015193 GSM7015193 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231081 GSM7015194_r1 SRP483237 GSE253061 SRS16635707 GSM7015194 GSM7015194 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231082 GSM7015195_r1 SRP483237 GSE253061 SRS16635708 GSM7015195 GSM7015195 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231083 GSM7015196_r1 SRP483237 GSE253061 SRS16635709 GSM7015196 GSM7015196 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231084 GSM7015197_r1 SRP483237 GSE253061 SRS16635710 GSM7015197 GSM7015197 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231085 GSM7015198_r1 SRP483237 GSE253061 SRS16635711 GSM7015198 GSM7015198 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231086 GSM7015199_r1 SRP483237 GSE253061 SRS16635712 GSM7015199 GSM7015199 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231087 GSM7015200_r1 SRP483237 GSE253061 SRS16635713 GSM7015200 GSM7015200 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231088 GSM7015201_r1 SRP483237 GSE253061 SRS16635714 GSM7015201 GSM7015201 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231089 GSM7015209_r1 SRP483237 GSE253061 SRS16635715 GSM7015209 GSM7015209 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231090 GSM7015210_r1 SRP483237 GSE253061 SRS16635717 GSM7015210 GSM7015210 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231091 GSM7015211_r1 SRP483237 GSE253061 SRS16635716 GSM7015211 GSM7015211 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231092 GSM7015212_r1 SRP483237 GSE253061 SRS16635719 GSM7015212 GSM7015212 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231093 GSM7015213_r1 SRP483237 GSE253061 SRS16635718 GSM7015213 GSM7015213 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231094 GSM7015214_r1 SRP483237 GSE253061 SRS16635720 GSM7015214 GSM7015214 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231095 GSM7015215_r1 SRP483237 GSE253061 SRS16635721 GSM7015215 GSM7015215 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231096 GSM7015216_r1 SRP483237 GSE253061 SRS16635722 GSM7015216 GSM7015216 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231097 GSM7015231_r1 SRP483237 GSE253061 SRS16635723 GSM7015231 GSM7015231 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231098 GSM7015232_r1 SRP483237 GSE253061 SRS16635724 GSM7015232 GSM7015232 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231099 GSM7015233_r1 SRP483237 GSE253061 SRS16635725 GSM7015233 GSM7015233 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231100 GSM7015234_r1 SRP483237 GSE253061 SRS16635726 GSM7015234 GSM7015234 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231101 GSM7015235_r1 SRP483237 GSE253061 SRS16635727 GSM7015235 GSM7015235 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231102 GSM7015236_r1 SRP483237 GSE253061 SRS16635728 GSM7015236 GSM7015236 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231103 GSM7015237_r1 SRP483237 GSE253061 SRS16635730 GSM7015237 GSM7015237 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231104 GSM7015245_r1 SRP483237 GSE253061 SRS16635729 GSM7015245 GSM7015245 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231105 GSM7015246_r1 SRP483237 GSE253061 SRS16635731 GSM7015246 GSM7015246 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231106 GSM7015247_r1 SRP483237 GSE253061 SRS16635732 GSM7015247 GSM7015247 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231107 GSM7015248_r1 SRP483237 GSE253061 SRS16635733 GSM7015248 GSM7015248 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231108 GSM7015249_r1 SRP483237 GSE253061 SRS16635734 GSM7015249 GSM7015249 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231109 GSM7015250_r1 SRP483237 GSE253061 SRS16635735 GSM7015250 GSM7015250 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231110 GSM7015251_r1 SRP483237 GSE253061 SRS16635736 GSM7015251 GSM7015251 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231111 GSM7015252_r1 SRP483237 GSE253061 SRS16635737 GSM7015252 GSM7015252 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231115 GSM7015121_r1 SRP483237 GSE253061 SRS16635740 GSM7015121 GSM7015121 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231116 GSM7015122_r1 SRP483237 GSE253061 SRS16635742 GSM7015122 GSM7015122 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231117 GSM7015130_r1 SRP483237 GSE253061 SRS16635743 GSM7015130 GSM7015130 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231118 GSM7015131_r1 SRP483237 GSE253061 SRS16635744 GSM7015131 GSM7015131 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231119 GSM7015132_r1 SRP483237 GSE253061 SRS16635745 GSM7015132 GSM7015132 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231120 GSM7015133_r1 SRP483237 GSE253061 SRS16635746 GSM7015133 GSM7015133 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231121 GSM7015134_r1 SRP483237 GSE253061 SRS16635747 GSM7015134 GSM7015134 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231122 GSM7015135_r1 SRP483237 GSE253061 SRS16635748 GSM7015135 GSM7015135 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231123 GSM7015136_r1 SRP483237 GSE253061 SRS16635749 GSM7015136 GSM7015136 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231124 GSM7015164_r1 SRP483237 GSE253061 SRS16635750 GSM7015164 GSM7015164 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231125 GSM7015165_r1 SRP483237 GSE253061 SRS16635751 GSM7015165 GSM7015165 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231126 GSM7015173_r1 SRP483237 GSE253061 SRS16635752 GSM7015173 GSM7015173 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231127 GSM7015174_r1 SRP483237 GSE253061 SRS16635753 GSM7015174 GSM7015174 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231128 GSM7015175_r1 SRP483237 GSE253061 SRS16635754 GSM7015175 GSM7015175 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231129 GSM7015176_r1 SRP483237 GSE253061 SRS16635755 GSM7015176 GSM7015176 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231130 GSM7015177_r1 SRP483237 GSE253061 SRS16635756 GSM7015177 GSM7015177 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231131 GSM7015178_r1 SRP483237 GSE253061 SRS16635757 GSM7015178 GSM7015178 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231132 GSM7015179_r1 SRP483237 GSE253061 SRS16635758 GSM7015179 GSM7015179 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231133 GSM7015180_r1 SRP483237 GSE253061 SRS16635759 GSM7015180 GSM7015180 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231134 GSM7015101_r1 SRP483237 GSE253061 SRS16635760 GSM7015101 GSM7015101 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231135 GSM7015102_r1 SRP483237 GSE253061 SRS16635761 GSM7015102 GSM7015102 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231136 GSM7015103_r1 SRP483237 GSE253061 SRS16635762 GSM7015103 GSM7015103 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231137 GSM7015104_r1 SRP483237 GSE253061 SRS16635763 GSM7015104 GSM7015104 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231138 GSM7015105_r1 SRP483237 GSE253061 SRS16635764 GSM7015105 GSM7015105 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231139 GSM7015106_r1 SRP483237 GSE253061 SRS16635765 GSM7015106 GSM7015106 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231140 GSM7015107_r1 SRP483237 GSE253061 SRS16635766 GSM7015107 GSM7015107 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231141 GSM7015108_r1 SRP483237 GSE253061 SRS16635767 GSM7015108 GSM7015108 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231142 GSM7015145_r1 SRP483237 GSE253061 SRS16635768 GSM7015145 GSM7015145 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231143 GSM7015146_r1 SRP483237 GSE253061 SRS16635769 GSM7015146 GSM7015146 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231144 GSM7015147_r1 SRP483237 GSE253061 SRS16635770 GSM7015147 GSM7015147 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231145 GSM7015148_r1 SRP483237 GSE253061 SRS16635771 GSM7015148 GSM7015148 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231146 GSM7015149_r1 SRP483237 GSE253061 SRS16635772 GSM7015149 GSM7015149 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231147 GSM7015150_r1 SRP483237 GSE253061 SRS16635773 GSM7015150 GSM7015150 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231148 GSM7015151_r1 SRP483237 GSE253061 SRS16635774 GSM7015151 GSM7015151 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231149 GSM7015217_r1 SRP483237 GSE253061 SRS16635776 GSM7015217 GSM7015217 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231150 GSM7015218_r1 SRP483237 GSE253061 SRS16635775 GSM7015218 GSM7015218 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231151 GSM7015219_r1 SRP483237 GSE253061 SRS16635777 GSM7015219 GSM7015219 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231152 GSM7015220_r1 SRP483237 GSE253061 SRS16635778 GSM7015220 GSM7015220 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231153 GSM7015221_r1 SRP483237 GSE253061 SRS16635779 GSM7015221 GSM7015221 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231154 GSM7015222_r1 SRP483237 GSE253061 SRS16635780 GSM7015222 GSM7015222 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231155 GSM7015223_r1 SRP483237 GSE253061 SRS16635781 GSM7015223 GSM7015223 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231163 GSM7015159_r1 SRP483237 GSE253061 SRS16635789 GSM7015159 GSM7015159 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231164 GSM7015160_r1 SRP483237 GSE253061 SRS16635791 GSM7015160 GSM7015160 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231165 GSM7015161_r1 SRP483237 GSE253061 SRS16635790 GSM7015161 GSM7015161 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231166 GSM7015162_r1 SRP483237 GSE253061 SRS16635792 GSM7015162 GSM7015162 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231167 GSM7015163_r1 SRP483237 GSE253061 SRS16635793 GSM7015163 GSM7015163 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231483 GSM7015109_r1 SRP483237 GSE253061 SRS16636123 GSM7015109 GSM7015109 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231484 GSM7015110_r1 SRP483237 GSE253061 SRS16636124 GSM7015110 GSM7015110 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231485 GSM7015111_r1 SRP483237 GSE253061 SRS16636125 GSM7015111 GSM7015111 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231486 GSM7015112_r1 SRP483237 GSE253061 SRS16636126 GSM7015112 GSM7015112 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231487 GSM7015113_r1 SRP483237 GSE253061 SRS16636127 GSM7015113 GSM7015113 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231488 GSM7015114_r1 SRP483237 GSE253061 SRS16636128 GSM7015114 GSM7015114 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231489 GSM7015115_r1 SRP483237 GSE253061 SRS16636129 GSM7015115 GSM7015115 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231583 GSM7015123_r1 SRP483237 GSE253061 SRS16636223 GSM7015123 GSM7015123 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231584 GSM7015124_r1 SRP483237 GSE253061 SRS16636224 GSM7015124 GSM7015124 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231585 GSM7015125_r1 SRP483237 GSE253061 SRS16636225 GSM7015125 GSM7015125 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231586 GSM7015126_r1 SRP483237 GSE253061 SRS16636226 GSM7015126 GSM7015126 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231587 GSM7015127_r1 SRP483237 GSE253061 SRS16636227 GSM7015127 GSM7015127 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231777 GSM7014921_r1 SRP483237 GSE253061 SRS16636417 GSM7014921 GSM7014921 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231778 GSM7014922_r1 SRP483237 GSE253061 SRS16636418 GSM7014922 GSM7014922 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231779 GSM7014923_r1 SRP483237 GSE253061 SRS16636419 GSM7014923 GSM7014923 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231780 GSM7014924_r1 SRP483237 GSE253061 SRS16636420 GSM7014924 GSM7014924 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231781 GSM7014925_r1 SRP483237 GSE253061 SRS16636421 GSM7014925 GSM7014925 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231782 GSM7014926_r1 SRP483237 GSE253061 SRS16636422 GSM7014926 GSM7014926 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231783 GSM7014927_r1 SRP483237 GSE253061 SRS16636423 GSM7014927 GSM7014927 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231784 GSM7014928_r1 SRP483237 GSE253061 SRS16636424 GSM7014928 GSM7014928 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231786 GSM7014951_r1 SRP483237 GSE253061 SRS16636426 GSM7014951 GSM7014951 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231787 GSM7014952_r1 SRP483237 GSE253061 SRS16636427 GSM7014952 GSM7014952 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231788 GSM7014953_r1 SRP483237 GSE253061 SRS16636428 GSM7014953 GSM7014953 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231789 GSM7014954_r1 SRP483237 GSE253061 SRS16636429 GSM7014954 GSM7014954 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231790 GSM7014955_r1 SRP483237 GSE253061 SRS16636430 GSM7014955 GSM7014955 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231791 GSM7014956_r1 SRP483237 GSE253061 SRS16636431 GSM7014956 GSM7014956 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231792 GSM7014966_r1 SRP483237 GSE253061 SRS16636432 GSM7014966 GSM7014966 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231793 GSM7014967_r1 SRP483237 GSE253061 SRS16636433 GSM7014967 GSM7014967 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231794 GSM7014968_r1 SRP483237 GSE253061 SRS16636434 GSM7014968 GSM7014968 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231795 GSM7014969_r1 SRP483237 GSE253061 SRS16636435 GSM7014969 GSM7014969 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231796 GSM7014970_r1 SRP483237 GSE253061 SRS16636436 GSM7014970 GSM7014970 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231797 GSM7014971_r1 SRP483237 GSE253061 SRS16636437 GSM7014971 GSM7014971 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231798 GSM7014979_r1 SRP483237 GSE253061 SRS16636438 GSM7014979 GSM7014979 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231799 GSM7014980_r1 SRP483237 GSE253061 SRS16636439 GSM7014980 GSM7014980 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231800 GSM7014981_r1 SRP483237 GSE253061 SRS16636440 GSM7014981 GSM7014981 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231801 GSM7014982_r1 SRP483237 GSE253061 SRS16636441 GSM7014982 GSM7014982 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231820 GSM7014965_r1 SRP483237 GSE253061 SRS16636459 GSM7014965 GSM7014965 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231890 GSM7015079_r1 SRP483237 GSE253061 SRS16636530 GSM7015079 GSM7015079 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231891 GSM7015116_r1 SRP483237 GSE253061 SRS16636531 GSM7015116 GSM7015116 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231892 GSM7015117_r1 SRP483237 GSE253061 SRS16636532 GSM7015117 GSM7015117 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231893 GSM7015118_r1 SRP483237 GSE253061 SRS16636533 GSM7015118 GSM7015118 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231894 GSM7015119_r1 SRP483237 GSE253061 SRS16636534 GSM7015119 GSM7015119 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231895 GSM7015120_r1 SRP483237 GSE253061 SRS16636535 GSM7015120 GSM7015120 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550 SRX19231974 GSM7014929_r1 SRP483237 GSE253061 SRS16636614 GSM7014929 GSM7014929 RNA-Seq TRANSCRIPTOMIC cDNA Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina). NextSeq 550