GSM9024542: Sham_1, scRNA-seq; Mus musculus; RNA-Seq
Mice subjected BDL and Sham operation were first anesthetized. After cannulation of the inferior vena cava, Hanks' balanced salt solution (HBSS) without Ca2+ and Mg2+ containing 1% EDTA was used to perfuse the liver in situ retrograde perfusion through the portal vein for 5 min at 37°C. Then, NCD mice received a retrograde stepwise perfusion of HBSS with Ca2+ and Mg2+ containing 0.5 mg/mL collagenase Ⅳfor 10 min at 37°C, while CDAHFD mice and BDL mice received the same perfusion solution with a higher concentration of collagenase Ⅳ at 1 mg/mL. After perfusion, the liver was rapidly collected and carefully dispersed into ice-cold HBSS without Ca2+ and Mg2+. After removing as much liver parenchyma as possible, the minced bilio-vascular tree fragments were incubated in low glucose DMEM containing 0.075 mg/mL collagenase P, 0.02 mg/mL DNase, 3% FBS, 1% Hepes and 1% Penicillin-Streptomycin at 37°C for 15 min under agitation. The bilio-vascular fragments were then filtered and collected using a 350-mesh (40 µm) cell sieve. Following centrifugation at 1,250 rpm for 5 min at 4°C, the cell pellet was resuspended and incubated with 0.05% Trypsin-EDTA containing 0.02 mg/mL DNase for 15 min at 37°C under agitation. Next, the dissociated cells were gently and repeatedly pipetted and filtered three times through a 500-mesh (25 µm) cell sieve to obtain single-cell suspensions. Finally, the dead cells were removed from single-cell suspensions with the Dead Cell Removal kit according to the manufacturer's protocol. Cell viability (> 95%) was determined using trypan blue staining. 10X Genomics Chromium Single-Cell 3'kits (V3) were used to prepare barcoded scRNA-seq libraries according to the manufacturer's protocol. Single-cell suspensions were loaded onto 10X Chromium Single-Cell Controller Instrument to generated single cell gel beads in emulsions (GEMs), aiming to capture a total of 2,000-4,000 cells per library for Control samples (Sham mice), and 8,000-10,000 cells per library for BDL samples. After GEMs generation, DNA amplification and library construction were performed, and the libraries were sequenced on a Novaseq 6000 (Illumina) platform (paired-end multiplexing run,150 bp).