GSM6203035: EMB, scRNAseq; Homo sapiens; RNA-Seq
Biliary organoids  were spun down and resuspended in 0.25% trypsin and incubated for 30 minutes at 37 °C. At the end of the incubation, organoids were mechanically dissociated by pipetting. Trypsin digestion was blocked by addition of ice-cold resuspension buffer (2% FBS, 1.25 mM CaCl2, 4 mM MgCl2, 10mM HEPES, 5 mM glucose in 1X HBSS) and cells were recovered by centrifugation (1200 rpm for 4 minutes). Cells were resuspended in resuspension buffer, filtered through a 35 µm filter (Corning), counted and checked for viability using Countess II automatic cell counter (Thermo Fisher Scientific). Cells were recovered by centrifugation (1500 rpm for 5 minutes) and resuspended at the desired concentration in PBS containing 0.04% bovine serum albumin. For 10X Genomics, single cell RNA sequencing 10,000 cells were loaded onto 10X Genomics Single Cell 3' Chips along with maaster mix as per manufacturer's instructions. Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The resulting libraries were sequenced on a NovaSeq6000.