SRX12777017 GSM5656659 SRP343185 SRS10722165 GSM5656659 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq NextSeq 500 gds 305656659 GSM5656659 GEO Accession GSM5656659 SRX12777018 GSM5656660 SRP343185 SRS10722166 GSM5656660 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq NextSeq 500 gds 305656660 GSM5656660 GEO Accession GSM5656660 SRX12777019 GSM5656661 SRP343185 SRS10722167 GSM5656661 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq Illumina NovaSeq 6000 gds 305656661 GSM5656661 GEO Accession GSM5656661 SRX12777020 GSM5656662 SRP343185 SRS10722168 GSM5656662 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq Illumina NovaSeq 6000 gds 305656662 GSM5656662 GEO Accession GSM5656662 SRX12777021 GSM5656663 SRP343185 SRS10722169 GSM5656663 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq Illumina NovaSeq 6000 gds 305656663 GSM5656663 GEO Accession GSM5656663 SRX12777022 GSM5656664 SRP343185 SRS10722170 GSM5656664 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq Illumina NovaSeq 6000 gds 305656664 GSM5656664 GEO Accession GSM5656664 SRX12777023 GSM5656665 SRP343185 SRS10722171 GSM5656665 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq Illumina NovaSeq 6000 gds 305656665 GSM5656665 GEO Accession GSM5656665 SRX12777024 GSM5656666 SRP343185 SRS10722172 GSM5656666 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq Illumina NovaSeq 6000 gds 305656666 GSM5656666 GEO Accession GSM5656666 SRX12777025 GSM5656667 SRP343185 SRS10722173 GSM5656667 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq Illumina NovaSeq 6000 gds 305656667 GSM5656667 GEO Accession GSM5656667 SRX12777026 GSM5656668 SRP343185 SRS10722174 GSM5656668 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq Illumina NovaSeq 6000 gds 305656668 GSM5656668 GEO Accession GSM5656668 SRX12777027 GSM5656669 SRP343185 SRS10722175 GSM5656669 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq Illumina NovaSeq 6000 gds 305656669 GSM5656669 GEO Accession GSM5656669 SRX12777028 GSM5656670 SRP343185 SRS10722176 GSM5656670 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq Illumina NovaSeq 6000 gds 305656670 GSM5656670 GEO Accession GSM5656670 SRX12777029 GSM5656671 SRP343185 SRS10722177 GSM5656671 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq Illumina NovaSeq 6000 gds 305656671 GSM5656671 GEO Accession GSM5656671 SRX12777030 GSM5656672 SRP343185 SRS10722178 GSM5656672 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq Illumina NovaSeq 6000 gds 305656672 GSM5656672 GEO Accession GSM5656672 SRX12777031 GSM5656673 SRP343185 SRS10722179 GSM5656673 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq Illumina NovaSeq 6000 gds 305656673 GSM5656673 GEO Accession GSM5656673 SRX12777032 GSM5656674 SRP343185 SRS10722180 GSM5656674 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq Illumina NovaSeq 6000 gds 305656674 GSM5656674 GEO Accession GSM5656674 SRX12777033 GSM5656675 SRP343185 SRS10722181 GSM5656675 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq Illumina NovaSeq 6000 gds 305656675 GSM5656675 GEO Accession GSM5656675 SRX12777034 GSM5656676 SRP343185 SRS10722182 GSM5656676 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq Illumina NovaSeq 6000 gds 305656676 GSM5656676 GEO Accession GSM5656676 SRX12777035 GSM5656677 SRP343185 SRS10722183 GSM5656677 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq Illumina NovaSeq 6000 gds 305656677 GSM5656677 GEO Accession GSM5656677 SRX12777036 GSM5656678 SRP343185 SRS10722184 GSM5656678 RNA-Seq TRANSCRIPTOMIC cDNA [in vivo scRNAseq] Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation. Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration. Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [in vitro scRNAseq] Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS. [scRNAseq] Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500. [bulkRNAseq] To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11 (D2, D5, D7, D10, D11) of differentiation on Transwells. N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer's protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN). scRNAseq & BulkRNAseq Illumina NovaSeq 6000 gds 305656678 GSM5656678 GEO Accession GSM5656678