GSM9228384: CD45-EPCAM- stromal cells_110373; Homo sapiens; ATAC-seq
Surgical specimens were cleaned up by dissection, and the external muscle layer removed after whole thickness samples were taken for pathology/spatial work. Remaining mucosa and submucosa was minced using surgical scissors and scalpel blades, after removal of bulk epithelial cells by two washes in PBS containing antibiotics (penicillin/streptomycin, amphotericin B, gentamicin, ciprofloxacin) and 5 mM EDTA (Sigma-Aldrich). Minced tissue was then subjected to multiple rounds of digestion in RPMI 1640 medium, 5 mM HEPES, antibiotics as above and 1 mg/ml of Collagenase A and DNase I (Sigma-Aldrich). After 30 min, digestion supernatant containing cells was removed, filtered through a cell strainer, spun down and resuspended in 10 ml of PBS containing 5% bovine serum albumin (BSA) and 5 mM EDTA. Remaining tissue was then topped up with fresh digestion medium until no more cells were liberated. Single-cell suspensions obtained from tissue digests were blocked in PBS 5% BSA containing human anti-Fc block (Miltenyi) stained for FACS analysis or sorting with Fixable Viability Dye eFluor 780 (eBioscience, 1:1,000), DAPI (Invitrogen, 1:50,000) and antibodies (all from Biolegend, except anti-PDPN: clone NZ-1.3 from eBioscience) in PBS with 5% BSA and 5 mM EDTA for 20 min on ice. After washing in the same buffer, cells were sorted on a BD Aria III (100-µm nozzle) for the EPCAM-CD45- stromal fraction. Sorted fractions were immediately processed for 10x single-cell analysis. Samples were processed according to 10X Chromium Next GEM Single Cell ATAC kit manufacturer's instructions and sequenced on a NovaSeq 6000