GSM8412877: HC2 ileum; Homo sapiens; RNA-Seq
human intestinal biopsies were collected in RPMI and processed fresh to ontain single cell suspension. Biopsies underwent two 20 minute rounds of dissociation to remove the epithelial layer (Dissociation media: 10 ml HBSS (free of calcium and magnesium) EDTA (0.5M, pH 8, Invitrogen) and HEPES (1M, Lonza)) was followed by transfer to 10 ml of digestion solution (containing RPMI, FBS (Corning 35-010-CV), 0.005 g of Type IV collagenase (Sigma-Aldrich, cat # C5138-1G) and DNAase I (Sigma Aldrich Fine Chemicals Biosciences cat # 10104159001)) and incubated for 40min at 37°C using gentle agitation (180 rpm). The digested tissue was then further mechanically broken down by aspirating 10-times with a 10ml syringe by sequentially filtering through a 100-µm and a 40-µm cell strainer and washed with RPMI twice to obtain a single cell suspension of the intestinal lamina propria. For scRNASeq, Gene expression libraries were prepared as per 10x Genomics protocol. For RNA-seq Gene expression libraries were prepared as per SMART-Seq v4 Ultra Low Input Nextera XT or SureSelect XT RNA Direct v6 protocols