GSM5656659: sc Monolayers Pooled Conditions [GEX]; Homo sapiens; RNA-Seq
[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