GSM7747465: Wild-type (WT), scRNAseq; Mus musculus; RNA-Seq
P5 KO (Bap1 fl/fl; Tyr-Cre +; R26 TdT/TdT,  n=3 males) and WT (Bap1 wt/wt; Tyr-Cre +; R26 TdT/TdT, n=5 (3 males/ 2 females)) pups were euthanized and rapidly dissected on ice. Full-length colons were opened on the mesenteric side and the muscularis layer was removed using fine forceps. For each pup, muscularis tissue in carboxygenated (5% CO2/95% O2) 1× Hank's balanced salt solution (HBSS; Thermo-Fisher Scientific; Cat. #14025092) was cut into small pieces with insulin needles, and dissociated with Liberase (0.625 mg/mL; Sigma-Aldrich; Cat#:5401135001) plus DNase I (0.1 mg/mL; Millipore Sigma; Cat#:11284932001), MgCl2 (6 mM; Cat#:BP214-500), in DMEM/F-12 (Thermo-Fisher Scientific; Cat#:11320033) for 40 minutes at 37°C. Cells were triturated (P1000) every 10-15 minutes during Liberase digestion. Dissociated cells were passed through Falcon 35-μm filters (Corning, Corning, NY; Cat#:352235), pelleted (150 g, 5 minutes, 4°C) after addition of 0.04% w/v BSA (Thermo-Fisher Scientific; Cat#:AM2618) in HBSS (⅓ final volume), and then resuspended in w/v 1% BSA (Thermo-Fisher Scientific; Cat#:AM2618) in HBSS. After an additional pass through Falcon 35-μm filters, the cells were sorted (BD FACSJazz, 100 μm nozzle) into 300-μL 0.04% BSA in HBSS. Sorted cells were pelleted (150 g, 5 minutes, 4°C), supernatant was removed leaving only ~30 μL and the samples were submitted for single cell RNA sequencing. Libraries prepared with Chromium Single Cell 3′ Reagent Kits v3 (10x Genomics) according the manufacturer's instructions.