GSM8744529: Bcat_GOF_day4; Mus musculus; RNA-Seq
Briefly, freshly harvested intestine was flushed with cold PBS, cut into 2 cm open fragments, followed by two incubation steps under constant rotation with pre-digestion buffer (HBSS containing 10% FBS, 10 mM HEPES and 5 mM EDTA) at 37°C for 20 minutes twice. Next, tissues were rinsed with wash buffer (HBSS containing 2% FBS, 10 mM HEPES), and  incubated with pre-warmed digestion buffer (RPMI medium containing 10% FBS, 1% P/S, 15 mM HEPES, 25 U/mL of collagenase IV (Worthington LS004186), 100 U/mL of DNase I (Sigma D4513), 0.3 g/100 mL of Dispase II (Gibco 17105041)) and rotated at 37°C for 30 minutes. Suspension of digested tissue was vortexed vigorously for 20 seconds every 10 minutes to release single cells. Cell solution was passed through a 40 μm cell strainer and cells were pelleted by centrifugation at 400 g for 5 minutes at 4 °C and resuspended in RPMI medium containing 10% FBS. Library construction was performed per manufacturer's protocol using 10X Genomics Chromium Next GEM Single Cell 5' Kit V2