GSM7968133: WT-3, scRNAseq; Mus musculus; RNA-Seq
Briefly, livers from WT, CFTR-KO and CFTR-KO-GC mice were perfused with Hanks' A and then Hanks' B medium containing 0.05% collagenase (Roche Applied Science, Indianapolis, IN) and 0.8 units of trypsin inhibitor (Sigma-Aldrich, Saint Louis, MO) per unit of tryptic activity. The livers were excised and transferred into cold HBSS buffer on ice, the gallbladder was surgically removed, and the liver capsule was disrupted. Gentle mechanical agitation was applied to release most of the hepatocyte fraction, that was discarded. The hilum portion was removed, and the remaining tissue was subjected to a three-steps digestion with Collagenase Type IV (150 U/mL) for 30 mins at 37°C, Accutase (EMD Millipore) for 30 mins at 37°C and Trypsin (0.25%) for 20 minutes at 37°C. After each step, cells were collected and filtered with a 100 mm cell strainer, washed, and re-suspended in ice-cold resuspension buffer containing 2% FBS. After the last step, the single cell fractions were combined and cells were recovered by centrifugation (1200 rpm, 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 (1000/ml) were resuspended in PBS-0.04% BSA and submitted to the Yale Center for Genome Analysis for processing. 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.