GSM6593520: Ep+, replicate 1, scRNAseq; Mus musculus; RNA-Seq
Stomach tissues, obtained from Dox-treated or untreated mice, were minced with razor blades and incubated for 45 min at 37°C in HBSS (Gibco, #14025-092) supplemented with 3 mg/ml collagenase P (Roche, #11213865001), 1 mg/ml Dispase II (Roche, #04942078001), and 1 µg/ml DNase I (Roche, #10104159001). The dissociated cells were washed twice with FACS buffer [D-PBS(−) containing 0.5% BSA and 0.5 mM EDTA], and incubated with Accumax reagent (Innovative Cell Technologies, AM-105) for 15 min at 37°C. Subsequently, the cells were subjected to gentle pipetting and filtration with 100 µm filter and used for the following FACS experiments.  The enzymatically digested samples were subjected to antibody staining for 30 min on ice. Anti-mouse Cd31 antibody conjugated with APC (1:300; Biolegend, #102410), anti-mouse Cd45 antibody conjugated with APC (1:300; Biolegend, #103112), and anti-mouse Epcam antibody conjugated with PE (1:300; eBioscience, #12-5791-82) were used to stain the cells. After the immunostaining, the cells were subjected to Calcein Blue AM viability dye staining at 37°C for 15 min according to the manufacturer's protocol (Invitrogen, #65-0855-39). The resulting cells were collected by centrifugation, re-suspended with FACS buffer, added with TO-PRO3 Ready Flow reagent (Invitrogen, R37170), and subjected to sorting by FACS AriaIIIu cell sorter (Becton Dickinson). Briefly, the cells were subjected to sequential sorting with forward scatter (FSC-H vs. FSC-W), side scatter (SSC-H vs. SSC-W) to select single-cells. Subsequently, cells with poor membrane integrities, hematopoietic and endothelial cells were removed by negative selection against TO-PRO3, Cd45 and Cd31 signals, respectively. After selecting Calcein blue-positive viable cells, Epcam-positive cells (Ep+) and Epcam/GFP-positive cells (GFP+) were selected and analyzed for GFP+ ratio or sorted for single-cell RNA-seq. Libraries were constructed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, Ep+ and GFP+ single cells were prepared from the stomach of female H2B-GFP mice (n=2) at 6 weeks of post Dox treatment and FACS-sorted as described above. The cells were then loaded onto Chromium next GEM chip G (PN-2000177, 10x Genomics) and captured into Gel Beads-in-emulsion (GEMs) droplets using Chromium Controller (10x Genomics). cDNA synthesis and sample-indexed library construction were performed using Chromium next GEM single cell 3' reagent kit (PN-1000121, 10x Genomics) according to the manufacturer's protocol.