GSM9108421: Liver, FLC, rep3; Homo sapiens; RNA-Seq
Single nuclei were isolated from FLC tumors or matched surrounding liver tissue as previously described, with some modifications. Briefly, frozen tissue samples were briefly hand-thawed, washed in ice-cold PBS, and cut into small pieces using dissecting scissors. All subsequent steps were performed directly on wet ice. Tissue was homogenized to liberate nuclei in TST buffer47 with 20 strokes of Dounce A and B homogenizers, and adequate nuclei isolation was confirmed by trypan blue uptake in a small aliquot of lysate. Lysate was 40μm filtered and recovered in ST buffer47 containing 0.5% BSA. Nuclei were pelleted in a swinging rotor centrifuge at 500g for 5 minutes at 4°C. Supernatant was then decanted and nuclei gently resuspended in PBS (no Mg/Cl) containing 1% BSA and a 1:200 dilution of Protector RNase (Sigma Aldrich PN-3335399001) for counting. All subsequent processing and single-cell emulsion was per kit instructions, Chromium Next GEM Single Cell 3' Reagent Kits v3.1 revision D (10X genomics) on a 10X Chromium Controller. Sequencing libraries were generated per kit instructions, Chromium Next GEM Single Cell 3' Reagent Kits v3.1 revision D (10X genomics), with the only modification being cDNA (step 2.2) was amplified 2 cycles more than recommended to account for the lower cDNA yield from nuclei. Library size and quality was determined on an Agilent Tapestation 2200. All samples passing QC were submitted for deep sequencing by GeneWiz with a targeted read depth of 20,000 reads per cell.