GSM7785744: Control Stromal cells from 7045, scRNA-seq; Homo sapiens; RNA-Seq
Crypts were meticulously scraped off from lamina propria layer after incubation with 2 mM EDTA in 1 mL Chelation buffer for 15 min at 4°C with rotation. The epithelial cells were obtained from the isolated crypts after digestion in 1.5 mL TrypLE along with 0.5 U/mL DNAse I (Roche) for 30 min at 37°C with 800 rpm using a ThermoMixer. The lamina propria cells were obtained from the remaining lamina propria layer after digestion in 500 μL of stroma dissociation enzyme mix (comprising with 0.13 WU/mL of Liberase-TH, 0.5U/mL DNAse I in 1x HBSS) for 30 min at 37°C with 800 rpm using a ThermoMixer and subsequent supernatant collection for three cycles in total. The Dead Cell Removal kit was employed following the manufacturer's instructions. Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 were used for library construction according to the manufacture's protocol. Briefly, a total of 10,000 cells were combined with Master Mix and transferred to an assembled Chromium Next GEM Chip G, which was filled with Gel Beads or 50% Glycerol (VWR), and the setup was processed in a Chromium Controller. GEMs were incubated with Dynabeads Cleanup Mix. The resulting cDNA was then subjected to Pellet Cleanup with SPRIselect reagent. Purified cDNA sample obtained from Pellet Cleanup were subjected to Fragmentation, End Repair & A-tailing, followed by Double Sided Size Selection using SPRIselect.