GSM8079819: Liver_sAH102; Homo sapiens; RNA-Seq
Venous blood was procured into EDTA-coated tubes. Within one hour of collection, red blood cells were lysed using ammonium chloride buffer (1:20, BioLegend®) for 10 minutes and the resulting WBCs were washed twice with PBS. Thin (1cm) liver sections were taken from the right lobe of explanted livers. The tissues were coarsely minced before 1 wash with PBS to remove excess red blood cells and albumin. The washed tissues were suspended in a 0.1% collagenase/0.025% hyaluronidase solution and incubated at 37ᵒC with constant agitation for 30 minutes. After incubation, tissue homogenates were centrifuged at 50g for 2 minutes to pellet debris and the resulting supernatant was collected and washed with PBS. Using OptiPrep® and DMEM, the density of the cell pellet was adjusted to ρ=1.5 and floated through discontinuous layers of ρ=1.2 and PBS. Viable liver cells were collected from the ρ=1.2/PBS interface, washed with PBS. Liver cells (6 samples) and WBCs (7 samples) from AC and sAH patients were loaded in the lanes using 10X Genomics Chromium Next GEM Single Cell 3' Kit v3.1 with a single capture lane per sample targeting recovery of 8,000 cells. All subsequent steps of library preparation and quality control were performed according to the 10X Genomics Chromium Single Cell 3' Reagent Kits User Guide. Sequencing was performed on an Illumina NovaSeq S4 at GENEWIZ (Azenta US, Inc.).