GSM7140805: Fasted liver, scRNAseq; Mus musculus; RNA-Seq
Primary hepatocytes were isolated by the 2-step collagenase perfusion through the inferior vena cava. Mice were perfused with Liver Perfusion Medium (Gibco #17701038) followed by Liver Digest Medium (Gibco #17703034). The liver cell suspension was filtered through a 70 μM strainer. Equal amounts of Hepatocyte Wash Medium (Gibco #17704024) was added to the liver cell suspension and centrifuged at 50 g for 5 minutes to collect hepatocytes. After washes, primary hepatocytes were resuspended in GBSS (Sigma Aldrich #G9779) with 0.7% CHAPS  (Sigma Aldrich #C5070) and 0.2U/μL RNase inhibitor (Roche Diagnostics #3335399001), and kept on ice for 5 min. The samples were centrifuged at 500g for 5 minutes and further washed twice using PBS with 1% BSA and 0.2U/μL RNase inhibitor. The nuclear extraction efficiency was determined with trypan blue under a microscope. The isolated nuclei were resuspended in the wash buffer to around 1x106 nuclei/mL. Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics).