title: GSM8152628: MASH Regression 8w 1; Mus musculus; RNA-Seq
library_construction_protocol: For primary liver cell isolation, mice livers were perfused by cannulation through the inferior vena cava with 0.33mg/ml pronase (Roche, Indianapolis, IN), followed by 0.67mg/ml collagenase D (Roche, Indianapolis, IN), for 5-10 minutes before ex vivo digestion in a solution containing pronase, collagenase D and DNase1 (Roche, Indianapolis, IN) for 10min in a rotating incubator at 370C. The dissociated cells were strained through a 100μm mesh. The cell suspension was centrifuged at 50g for 1 minute to remove the hepatocytes, supernatant was collected and centrifuged at 800g for 7 minutes. Cell pellet washed with GBSS-B buffer at 700g for 7 minutes. Cells were finally isolated by Nycodenz gradient centrifugation at 2000g for 20 minutes. The Hepatic stellate cells (HSC) layer (upper layer) and the other non-parenchymal cells (NPC) layer (lower layer) were then collected and washed with GBSS-B at 800g for 7 minutes and processed. Total RNA was isolated using Rneasy column (Qiagen, Valencia, CA). 10X Genomics 3' Single Cell RNA-seq (Chromium Next GEM Single Cell 3′ GEM, Library & Gel Bead Kit v3.1) following manufactures protocol.