GSM6190844
Flash frozen brain tissues were homogenized in a Dounce homogenizer in Lysis Buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.025% NP-40), 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 oC. Nuclei were washed and filtered twice with Nuclei Wash buffer (1% BSA in PBS with 0.2 U μl–1 RNasin (Promega)). Nuclei pellets were resuspended in 500 μl nuclei wash buffer and 900 µl 1.8 M sucrose. This 1,400 μl mixture was carefully layered on top of 500 μl 1.8 M sucrose and centrifuged at 13,000g for 45 min at 4 oC to separate the nuclei from myelin debris. The nuclei pellet was resuspended in nuclei wash buffer at 1,000 nuclei μl–1 and filtered through a 40-μm FlowMi Cell Strainer. Chromium Single Cell 5' Reagent Kit (v2) single nucleus RNA sequencing