title: NextSeq 500 sequencing; Single-cell RNA-seq of primary human gastric interstitial cells of Cajal
library_construction_protocol: Human gastric tissue from the antrum and corpus of the greater curvature of the stomach was collected from laparoscopic sleeve gastrectomy resections. This study was approved by the Western Sydney University Human Research Ethics Committee, and all tissues were obtained after written informed consent was provided by the patients. Tissue samples were stored immediately after surgery in ice-cold Hank's Balanced Salt Solution (HBSS; -Ca2+ and -Mg2+) before immediate transport and laboratory processing. Samples were dissected so that only the smooth muscle, devoid of the submucosa and mucosa, was used for experiments. Gastric muscle tissue was washed in HBSS (-Ca2+ and -Mg2+) containing 1% Penicillin/Streptomycin and 0.1% fungizone followed by PBS (-Ca2+ and -Mg2+) to remove excess blood. Tissue was minced into 1-2 mm3 pieces and incubated (1 hour, 37°C) on a rotator in Dulbecco's Modified Eagle Medium (DMEM) containing 0.2% Type IV collagenase and 0.02% DNase I from bovine pancreas. Semi-digested tissue was washed through a fine wire mesh strainer with DMEM containing 2% fetal bovine serum (FBS) and filtered through a 100-µm cell strainer followed by a 40-μm cell strainer. The single cell suspension was centrifuged (500 g, 5 min, 4°C), and the cells washed with FACS buffer: 0.5% BSA in PBS (-Ca2+ and -Mg2+). Red blood cells were lysed with ACK Lysing Buffer (1 mL, 30-60 sec, RT). The cells were then washed with 5 mL PBS (-Ca2+ and -Mg2+), centrifuged (300 g, 5 min, 4°C), resuspended in FACS buffer and counted using a haemocytometer. Cell suspension aliquots were incubated in the dark with Human Fc Receptor Blocking Reagent and anti-KIT-APC antibody (20 min, 4°C), and then washed in FACS buffer and centrifuged (300 g, 5 min). Hematopoietic cells were labelled using FITC-conjugated anti-CD45 and anti-CD11b antibodies (20 min, 4°C) before being washed as above. All samples were resuspended in 500 µL DAPI solution and re-filtered through a 40-μm cell strainer. Flow cytometry gates were set on live DAPI- cells using unstained and single-stained cell populations prior to cell purification via FACS using a BD FACSAria III cell sorter. RNA was collected immediately after cell sorting. Cells were processed immediately after collection using a 10X Genomics single cell 3' mRNA-prep kit as per the manufacturer instructions. Library construction was performed using a 10X Genomics single cell 3' mRNA-prep kit as per the manufacturer instructions.