title: GSM3937758: sc2000; Mus musculus; RNA-Seq
library_construction_protocol: Hepatocytes were isolated by a two-step collagenase perfusion. Animals were anesthetized (fentanyl 50ug/kg, midazolam 5mg/kg, medetomidine 500ug/kg, i.p.), immobilized in a supine position and the liver and portal vein exposed. The portal vein was cannulated with a 22G catheter and perfusion at 4ml/min with the buffers allowed to run to waste through an incision in the inferior vena cava. The liver was perfused with 10 ml of HBSS (Mg2+, Ca2+ free,10mM HEPES, pH7 ) followed by 25ml of HBSS containing EDTA ( 10mM HEPES pH7, 5mM EDTA). The EDTA was removed from the liver by perfusion with 10 ml of HBSS followed by digestion with 25 ml of HBSS containing collagenase ( 10mM HEPES, 1mM CaCl2, 0.5mg/ml collagenase IV, 0.01mg/ml collagenase 1A (Sigma)). The liver was then removed and the cells released by cutting the capsule and gentle agitation of the digested liver in stop buffer (HBSS, 10mM HEPES pH7, 5mM EDTA, 10mM citrate, 1% FBS) and passed through a 70µm filter. The cell suspension was spun at 30G min to pellet most of the hepatocyte fraction, the supernatant collected and remnant cells pelleted at 250xG. This cell pellet was washed once in stop buffer then re-suspended in 20% isotonic Percoll and overlaid on a layer of 80% isotonic Percoll and spun at 500G for 10 minutes. The cells at the interface of the two Percoll layers were collected and washed in PBS, re-suspended in PBS and counted using a cell counter (BioRad TC 20). scRNA-seq libraries were prepared from 1000, 2000, 5000 and 10000 cells using the Chromium Single Cell 3' Library & Gel Bead Kit v3 (10xGenomics). Libraries were prepared according to the manufacturer's protocol.
attributes.tag: GEO Accession
attributes.value: GSM3937758