GSM5655496: Donor 1 pooled regions [CSP]; Homo sapiens; RNA-Seq
Full human intestinal tracts (duodenum through descending colon) were received from HonorBridge (formerly Carolina Donor Services). Intestines were transported on ice in University of Wisconsin Solution, and tissue dissection began at UNC Chapel Hill within eight hours of cross-clamping. Fat and connective tissue were trimmed, and intestines were measured and regions separated, with the proximal 20 cm deemed Duodenum, Jejunum and ileum evenly splitting the remaining small intestine, and colon split into equal thirds for ascending, transverse, then descending colon. Two 3x3 cm mucosectomies were isolated from the center of each region for dissociation.   Full mucosa pieces were incubated in 10 mM NAC (Sigma-Aldrich A9165) in dPBS (Gibco 14190-144) at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer consisting of 5.6 mmol/L Na2HPO4 (Sigma S7907), 8.0 mmol/L KH2PO4 (Sigma P5655), 96.2 mmol/L NaCl (Sigma S5886), 1.6 mmol/L KCl (Sigma P5405), 43.4 mmol/L Sucrose (Fisher BP 220-1), 54.9 mmol/L d-sorbitol (Fisher BP439-500), and 100 µmol/L Y27632 (Selleck Chemical, S6390) then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mmol/L EDTA (Corning 46-034-Cl) and 0.5mmol/L DTT (Fisher Scientific BP172-5), then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield shakes were pooled for colon crypts, and 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 (Sigma P5380) in dPBS + Y-27632 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 + Y-27632 then resuspended in Advanced DMEM/F12 (Gibco 12634-010) + 1% Bovine Serum Albumin (Fisher Scientific BP1600-1) + Y-27632 for staining with AnnexinV-APC (1:100, BioLegend 640920) and with one TotalSeq Anti-Human Hashtag Antibody per region (1:100, BioLegend B0251-B0256) to allow for tracking all six regions with a single library preparation. Cells were washed and resuspended in AdvDMEM + 1% BSA +Y-27632 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 (PN-100012). Sequencing was performed on an Illumina NextSeq 500. scRNAseq