title: GSM5067107: Isolated single nuclei from 7 pooled hippocampi of 9-month old WT mice; Mus musculus; RNA-Seq
library_construction_protocol: Frozen hippocampi from 7-8 mice of the same genotype were pooled as a single sample. Tissue was homogenized with a Dounce homogenizer in lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.005% NP-40 in nuclease free water), and incubated on ice for 15 min. The suspension was filtered through a 30-μm filter to remove debris and pelleted at 500g for 5 min at 4 ℃. Nuclei were washed and filtered twice with Nuclei Wash Buffer (1x PBS with 1.0% BSA and 0.2 U/uL RNasin Ribonuclease Inhibitors (Promega)). Nuclei pellets were resuspended in 500 μl Nuclei Wash Buffer, and mixed with 900 µl Sucrose Cushion Buffer (2.7 mL Nuclei PURE 2M Sucrose Cushion Solution (Sigma-Aldrich, Cat# S9308) + 300 uL Nuclei PURE Sucrose Cushion Solution (Sigma-Aldrich, Cat# S9058)). The 1,400 μl mixture was carefully layered on top of 500 μl Sucrose Cushion Buffer, and centrifuged at 13,000g for 45 min at 4 ℃ to separate the nuclei from cell debris. The nuclei pellet was resuspended in Nuclei Wash Buffer and filtered through a 40-μm FlowMi Cell Strainer (Sigma-Aldrich, Cat# BAH136800040). Isolated single nuclei were subjected to droplet-based 3' end massively parallel single-cell RNA sequencing using Chromium Single Cell 3' Reagent Kits as per manufacturer's instructions (10x Genomics). The generated scRNAseq libraries were sequenced using an Illumina sequencer.