GSM8472691: S1, scRNAseq; Mus musculus; RNA-Seq
Primary hepatocytes were isolated by digestion of the liver with a modified two-step collagenase perfusion method. Briefly, the liver was perfused via superior vena cava with Ca-free Earles' solution (EBSS) containing 0.5 mM EGTA, followed by perfusion with Ca-containing EBSS with 0.3-0.4 mg/ml collagenase type IV (Sigma). Donor Fah+/+ primary hepatocytes were puriﬁed with Percoll (Sigma) (containing Percoll/10x EBSS, 9:1) and collected by a series of centrifugation steps (3 × 4min × 50g ). Purified liver cells need to be filtered through a 40um filter membrane. Only cell isolations with >95% viability were used for transplantation. Puriﬁed hepatocytes were diluted to 107 cells/ml in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 10 mM HEPES kept on ice Single-cell capturing and library constructions were carried out using Chromium Single Cell 3g Reagent kits v.2 and kit v.3 (10x Genomics) as well as Chromium Single Cell V(D)J Reagent kits (10xGenomics) for TILs according to the manufacturer's protocol. In brief, single cell suspension, barcoded gel beads and partitioning oil were loaded onto Chromium Chip A/B to obtain single-cell gel bead-in-emulsion. And then the polyadenylated transcripts were reverse-transcribed in each gel bead-in-emulsion.