GSM6556456: Human NASH Liver 3 R4C1, snRNA-seq; Homo sapiens; RNA-Seq
frozen human liver tissue (~60 mm3) was lysed by Dounce homogenization (Kimble 2mL: 20 times with Pestle A over ~60 seconds) in 2mL lysis buffer (10mM Tris pH 7.0, 10mM NaCl, 3mM MgCl2, 0.05% Triton X-100, 0.13% RNAse Inhibitor (Enzymatics Y9240L, MA, USA), 0.25% Superase RNAse Inhibitor (Thermo Fisher Scientific AM2694, MA, USA)) with 45mM Actinomycin D, then dripped through a 40mm cell strainer (Celltreat 229481, MA, USA) into a protein low-binding 15mL centrifuge tube and the filter was washed into the collection tube with another 2mL of lysis buffer. After 5 minutes total exposure time to lysis buffer, samples were spun for 2 minutes at 300xg at 4°C; the supernatant was aspirated from the top first to remove fat, then the nuclei-containing pellet was resuspended in 5mL of wash buffer (DPBS + 2% BSA + 0.13% Enzymatics RNAse Inhibitor + 0.25% Superase RNAse Inhibitor + 3mM Actinomycin D) and filtered again through a 35mm strainer into a 5mL FACS tube. Nuclei were sorted into a 1.7mL a protein low-binding microcentrifuge tube pre-coated with 900mL of wash buffer on a Biorad S3e FACS using FSC/SSC > FSC-H vs FSC-W > FL4 vs. SSC gating to eliminate debris. Nuclei were pelleted at 300xg for 3 minutes at 4°C in a swing-bucket centrifuge and resuspended in wash buffer for 10x capture using an LT200 pipettor.  Nuclei preparations were processed by the Chromium 3′ Gene Expression V3 Kit according to the manufacturer's guidelines. Qubit 3 (Fisher Scientific) and 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) were used for quality check of cDNA.