GSM9168816: Liver, 6wk FAT-MASH Regression; Mus musculus; RNA-Seq
40mg of liver tissue each from 5 mice were pooled and stored in RPMI (Corning, 15-040-CM) + 10% fetal bovine serum (FBS; Invitrogen, 10082147) on ice overnight. The following morning, livers were injected with digestion buffer consisting of RPMI with 10% FBS, 0.25mL Collagenase IV (Sigma, C5138), and 0.1mg/mL DNase I (Sigma, D5025). For digestion, samples were initially minced and incubated at 37°C for 1 hour under slow agitation (80rpm), and then mechanically digested using 16G needles, before being filtered through 100uM and 70uM strainers. Thereafter, for gradient separation, samples were centrifuged (500xg, 4°C, 10 min), resuspended in 25% Percoll, and layered atop 70% Percoll (Cytiva, 17-0891-02), and centrifuged again (500xg, 20°C, 25 min, no acceleration/brake). Non-parenchymal cells (NPCs) were collected at the meeting point of the Percoll layers. NPCs were rinsed with 1XHBSS (no ions; Corning, 21-022-CV) and subjected to RBC lysis (Fisher, A1049201). A fraction of cells from this suspension was transferred to the Mount Sinai Genomics CoRE for counting, quality control and sequencing. Nuclei preparations were processed by the Chromium 3′ Single Cell 3' Reagent Kit according to the manufacturer's guidelines.