GSM7667335: MacControl_02; Mus musculus; RNA-Seq
Healthy and fibrotic murine liver were perfused with 1X PBS, cut into small pieces and digested in collagenase II (5mg/mL, Worthington Biochemical Corporation) at 37 oC for 2h with shaking. Digested cells were filtered through 40 μm nylon mesh and lysed with ACK lysis buffer (Quality Biological) for 10 min. After spinning, the cells were resuspended into 1X PBS. Hepatocytes were removed by spinning at 50xg for 10 min. The supernatant was further spined at 650xg for 3 min to get the nonparenchymal cells, which was further stained with CD45+ (BV785, Cat#103149, Biolegend) and F4/80+ (BV421, Cat#123132, Biolegend) for 30 min. Cells were further suspended in 1X PBS with 0.1% Draq7 live/dead staining. CD45+/F4/80+ macrophage was further sorted with BD FACSAria Fusion Sorter (BD Biosciences). RNA extracted and amplified (10 ng RNA/sample) using SMART-Seq® HT kit (Takara Bio, #634455) according to the manufacture's instruction PolyA-selected RNA was used to generate libraries using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB) according to the manufacturer's instructions