GSM9168823: Liver, Untreated Control [snRNA-Seq]; Mus musculus; RNA-Seq
40 to 60 mg of total liver tissue (pooled from 3 mice per condition) were chopped on ice in 1 mL of 0.03% TST buffer [146 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM CaCl2, and 21 mM MgCl2 with freshly added 0.03% Tween 20, 0.01% BSA, and RiboLock RNase Inhibitor (0.4 U/mL; Thermo Fisher Scientific, FEREO0382)] with Tungsten Noyes Spring Scissors (FST, 15514-12) following our previously published procedures. The resulting nuclei suspension was then filtered through 40μm cell strainers (Thermo Fisher Scientific, 352340) into a fresh 50-mL conical tube on ice. One more milliliter of 0.03% TST was used to rinse the cell strainer, and 3 mL of ST buffer were added to the nuclei suspension, followed by briefly mixing by gentle flicking. The nuclei suspensions were spun at 500xg for 5 min at 4°C in a swing-bucket centrifuge. The final nuclei pellet was suspended in 200μL of 0.4% BSA in PBS by pipetting 30 times with a regular p1000 tip. The resulting pellet was resuspended by pipetting 30 times in 200µL of 0.4% BSA/PBS, and nuclei were visually inspected using a 1:1 trypan blue solution under a light microscope. Samples were transferred to the Mount Sinai Genomics CoRE for counting, quality control and sequencing. Nuclei preparations were processed by the Chromium 3′ Gene Expression V2 Kit according to the manufacturer's guidelines.