GSM9652272: Ethanol + LPS Mouse Liver Resident Immune Cells Sample 1; Mus musculus; RNA-Seq
Liver and peripheral blood were harvested at sacrifice. For single-cell RNA sequencing (scRNA-seq), liver perfusion was performed via the portal vein using established protocols. Hepatic mononuclear cells were isolated by Percoll gradient centrifugation and enriched for CD45⁺ immune cells using magnetic-activated cell sorting (MACS; Miltenyi Biotec, Cat#130-052-301). LSEC enrichment was performed on CD45- fraction with CD146+ MACS beads (Miltenyi Biotec, Cat#130-092-007). CD45+ immune cells and LSECs were sorted by flow cytometry cell sorting for CD45+ or CD146+ cells (BD Biosciences FACS Aria III cell sorter). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient centrifugation from heparinized blood, then sorted for CD45+ cells on the same cell sorter. For intrahepatic cytometry experiments, liver perfusion, Percoll gradient centrifugation, and MACS beads enrichment steps are as described above. Enriched CD45+ cells were then processed for flow cytometry staining as described below. 90,000 CD45⁺ immune cells and 10,000 sorted liver sinusoidal endothelial cells (LSECs) were combined and processed using the 10x Genomics Chromium platform following manufacturer's protocols. Single-cell suspensions were subjected to library preparation and sequencing at the Mayo Clinic Genomic Core.