GSM8136431: Hippocampus, Control rep1; Mus musculus; RNA-Seq
Mouse hippocampal tissues were rapidly dissected in ice-cold homogenization buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH, pH=7.8). The tissue was homogenized using a loose-fitting pestle in 1.5 ml of homogenization buffer supplemented with 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, 1×EDTA-free protease inhibitor (Roche), and 60 U/mL RNasin Plus RNase Inhibitor (Promega, N2611). A 6% IGEPAL CA-630 solution was added to bring the homogenate to 0.3% IGEPAL CA-630, and the homogenate was further homogenized with five strokes of a tight-fitting pestle. The sample was filtered through a 50 μm filter, underlayed with solutions of 30% and 40% iodixanol (Sigma, D1556) in homogenization buffer, and centrifuged at 10,000 g for 20 min in a swinging bucket centrifuge at 4 °C. Nuclei were collected at the 30-40% interface, diluted with two volumes of homogenization buffer and concentrated by centrifugation for 10 min at 500 g at 4 °C. These nuclei can be used for snRNA sequencing. 10x Genomics Chromium single cell kit snRNA-seq