GSM9067565: HFD; Mus musculus; RNA-Seq
Primary hepatocytes and non-parenchymal liver cells were isolated using a two-step in situ perfusion method. Briefly, mice were anesthetized and subjected to portal vein cannulation for liver perfusion. The liver was first perfused with pre-warmed (37°C) calcium-free HBSS containing 0.5 mM EGTA for 5 minutes to disrupt tight junctions, followed by perfusion with HBSS containing 0.05–0.1% collagenase IV and 5 mM CaCl₂ for 10–12 minutes until the liver became visibly swollen and soft. The liver was then excised, minced, and filtered through 100 μm and 70 μm strainers. The cell suspension was centrifuged at 50 × g for 3 minutes at 4°C to pellet hepatocytes. The supernatant, containing non-parenchymal cells (NPCs), was collected and further purified by 40% Percoll density gradient centrifugation at 800 × g for 20 minutes without brake. Isolated cells were kept on ice throughout the procedure unless otherwise indicated, and viability was assessed by trypan blue exclusion. After cell counting, hepatocytes and non-parenchymal cells were mixed at a 1:1 ratio to generate a final cell suspension, which was used for single-cell RNA-seq library construction and sequencing. Single-cell RNA-seq libraries were generated using the 10x Genomics Chromium Next GEM Single Cell 3' Kit v3.1. Cell suspensions (700–1200 cells/μL), barcoded gel beads, and partitioning oil were loaded into separate lanes of a Chromium Chip G to form Gel Beads-in-Emulsions (GEMs). Most GEMs contained either no cell or a single cell. Inside each GEM, gel beads dissolved, releasing barcoded oligos for reverse transcription of mRNA from lysed cells. After GEMs were broken, cDNA was amplified, purified, and used for library construction, including fragmentation, end-repair, A-tailing, adaptor ligation, and PCR enrichment. The final library contained a 16 bp cell barcode, 12 bp UMI, and cDNA insert flanked by Illumina adapters. Sequencing was performed using paired-end reads (Read 1: 28 bp for barcode and UMI; Read 2: 91 bp for transcript information) on an Illumina platform.