GSM8720590: Demye2+Ctrl IgG2a; Mus musculus; RNA-Seq
Isolation of nuclei from the frozen brain tissue of the aforementioned groups was performed similarly as previously described. In brief, the flash frozen cortex and white matter were Dounce homogenized in 5 ml of ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.025% NP-40) for 15 min. The homogenates was filtered through a 30-μm cell filter and pelleted at 500g for 5 min at 4 °C. Nuclei pellet was then washed and filtered twice with Nuclei Wash buffer (1% BSA in PBS with 0.2 U/μl RNasin (Promega)), and were resuspended in 500 μl nuclei wash buffer and 900 µl 1.8 M sucrose. The total 1400 μl mixture was carefully layered on top of 500 μl 1.8 M sucrose and centrifuged at 13,000g for 45 min at 4 ℃ to separate the nuclei from myelin debris. Finally, the nuclei pellet was resuspended in nuclei wash buffer at 1,200 nuclei/μl and filtered through a 40-μm FlowMi Cell Strainer. The isolated mouse brain nuclei were subjected to droplet-based single-nucleus RNA sequencing (snRNA-seq) using the Chromium Single Cell 5′ Reagent Kits (v1) according to the manufacturer's instructions (10x Genomics).