GSM8679420: Control Heps; Mus musculus; RNA-Seq
In n=2 mice/group for each time point primary mouse hepatocytes and non-parenchymal cells (NPCs) were isolated by a multi-step ethylene glycol tetra-acetic acid (EGTA)/collagenase perfusion technique, through microcannulation of portal vein to ensure efficient perfusion of tissues. Cells were centrifuged at 50g x 3 minutes for three times to separate hepatocytes (on bottom) and NPCs (on top). Next, cell suspensions were FACS sorted by using FACSanto to remove dead cells and cellular debris, exploiting 7-Aminoactinomycin D (7-AAD) staining, thus allowing viable cell enrichment. CD45+positive cells were depleted by hepatocyte fractions. Hepatocytes and NPCs live cells were separately loaded on a Chromium Single Cell Instrument (10x Genomics) to generate single-cell gel beads in emulsion. RNA-seq libraries were prepared with the Chromium Single Cell Library protocol (10x Genomics) according to manufacturer's instructions, and sequencing was performed on the Novaseq machine using paired-end sequencing runs (3200 million paired-end 150bp) for gene expression analysis. Final libraries were checked with both Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and Agilent Bioanalyzer DNA assay or Caliper (PerkinElmer, Waltham, MA). Libraries were then prepared for sequencing and sequenced on paired-end 150 bp mode on NovaSeq 6000 (Illumina, San Diego, CA).