GSM9294407: GLP2-1 12M; Homo sapiens; RNA-Seq
balloon-assisted enteroscopy to obtain intestinal mucosa biopsy. The intestinal tissue was washed with Ca2+, Mg2+-free Hank's balanced salt solution (HBSS) (Nacalai Tesque, 17460-15) to remove fecal content. After washing, the intestinal tissue was incubated with HBSS containing 1 mM dithiothreitol (Invitrogen, 15508-013) and 1 mM EDTA (Nacalai Tesque, 06894-85) for 15 min at 37°C. The supernatant of the solution was harvested for epithelial cells by passing through a 100-μm cell strainer into a 50-mL Falcon tube. At the same time, the tissues without the epithelial cells were washed with HBSS and dissolved into solutions by incubation with HBSS containing 2.5% fetal bovine serum (FBS) (Thermo Fisher Scientific, 10270-106) and penicillin/streptomycin (Nacalai Tesque, 26253-84), 1.0 mg/mL collagenase Type3 (Worthington, 032-22364), and 0.1 mg/mL DNase (Sigma-Aldrich, DN25) for 60 min at 37 °C. The dissolved solution and digested tissues were homogenized by vigorous shaking and passed through a 100-m cell strainer into a 50-mL Falcon tube. The solution was centrifuged at 1,700 rpm for 5 min at 4°C , the supernatant was removed, and resuspended in RPMI-1640 (Nacalai Tesque, 00-5523-02) containing 10% FBS and penicillin/streptomycin (Nacalai Tesque, 26253-84) (16, 17). Isolated single cell suspensions of IE and LP cells were pooled and incubated with a Fixable Viability Dye eFluor (eBioscience, 65-0865-14) for 15 min at 4 °C. Live cells were sorted using BD FACS Aria-II (BD Bioscience) and loaded into Chromium Controller (10X Genomics). RNA-seq libraries were prepared using Chromium single-cell 5' gene expression library preparation (human) according to the manufacturer's instructions (10X Genomics). The libraries were then sequenced using a DNBSEQ (MGI Tech).