GSM7785727: Control colonoids from C9, GEX; Homo sapiens; RNA-Seq
Human colonoids were collected at day 10 after plating and digested with 0.05% trypsin for 10 minutes at 37°C in a bead bath (final ratio as 10% FBS was added to de-activate trypsin). Cells were further dissociated by pipetting up and down several times to get single cells. Following incubation with 1μg of TotalSeqtrade anti-human Hashtag Antibody (Hashtag 1 for untreated samples, Hashtag 2 for TNFα-treated samples,Hashtag 3 for IFNγ-treated samples,Hashtag 4 for TRAIL-treated samples) for 20min and DAPI for additional10 minutes on ice, DAPI- cells were sorted in 1xDPBS+0.04% BSA using a MoFlo Astrios sorter. Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 with Feature Barcoding technology for Cell Surface Protein (10X Genomics) were used for library construction according to the manufacture's protocol. The addition of Cell Hashing allowed for sample multiplexing. 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 (for 3ʹ Gene Expression library) and Supernatant Cleanup (for Cell Surface Protein library) 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.