GSM8208266: P30_PKCi; Mus musculus; RNA-Seq
Nuclei were isolated from flash-frozen mouse cerebral cortices collected at indicated postnatal days from indicated genotypes (6mm punch biopsies from n=3-5 mice of each genotype were pooled per sample). 15 ml of ice-cold nuclei homogenization buffer [2 M sucrose, 10 mM Hepes (pH 7.9), 25 mM KCl, 1 mM EDTA (pH 8.0), 10% glycerol, and freshly added Rnase inhibitors (80 U/ml)] were used to homogenize the tissues with Dounce tissue grinder (10 strokes with loose pestle and 10 strokes with tight pestle). Homogenates were transferred into an ultracentrifuge tube on top of 10 ml of fresh nuclei homogenization buffer and centrifuged at 24,000 rpm for 60 min at 4°C on a high-speed centrifuge. The nuclei pellet was resuspended in 1 ml of nuclei resuspension buffer [15 mM Hepes pH 7.4, 15 mM NaCl, 60 mM KCl, 2 mM MgCl2, 3 mM CaCl2, and freshly added Rnase inhibitors (80 U/ml)] and counted using a hemocytometer. The nuclei were centrifuged at 800 g for 10 min at 4°C and resuspended at a concentration of 500 to 1000 nuclei/μl for 10x Genomics Chromium loading. The single nucleus (sn)RNA-seq libraries were prepared by the Chromium Single Cell 3′ Reagent Kit v3.1 chemistry according to the manufacturer's instructions (10x Genomics) and sequenced using Illumina NovaSeq 6000 S4 at the Yale Center for Genome Analysis (YCGA).