GSM6862093: adult liver, scRNAseq; Mus musculus; RNA-Seq
Mouse primary hepatocytes were isolated by the two-step collagenase perfusion method, which was modified from a previous protocol (Charni-Natan and Goldstein, 2020). Briefly, mice were anesthetized with pentobarbital intraperitoneal (80 mg/kg body weight). The abdomen was then incised, and the liver is exposed with inferior vena cava and portal vein. The portal vein was cut and the liver was perfused with perfusion buffer (containing 0.5 mM EGTA) for 5-10 minutes followed by digestion buffer containing type I collagenase (Invitrogen) for about 10 minutes. The liver was then taken out and transferred into a petri dish filled with RPMI 1640 medium containing 2% FBS at 4°C. The liver was then torn gently to help the hepatocytes dissociation, and the cell suspension was filtered through a 70 μm cell strainer (BD Falcon), followed by centrifugation at 50 x g for 2 minutes at 4°C. Cells were in Percoll/RPMI 1640/10 x PBS (1:1:0.1) mixture and centrifuged at 300 x g for 5 min at 4°C. Purified hepatocytes were collected for single-cell RNA sequencing. cDNA library construction was performed using Single Cell 3′ Library Kit (v3.1 chemistry, 10x Genomics). Sequencing was performed on an Illumina HiSeq4000 platform (Illumina, one lane, 150 PE).