GSM8919350: Sample 12; Homo sapiens; RNA-Seq
Frozen human brain tissues were homogenized in a Dounce homogenizer using 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 minutes. The homogenate was filtered through a 30-μm mesh to remove debris and centrifuged at 500g for 5 minutes at 4°C to pellet the nuclei. Nuclei were then washed and filtered twice using Nuclei Wash Buffer (1% BSA in PBS with 0.2 U/μl RNasin, Promega). For myelin debris removal, the nuclei were resuspended in 500 μl of Nuclei Wash Buffer, mixed with 900 μl of 1.8 M sucrose, and carefully layered over 500 μl of 1.8 M sucrose. This gradient was centrifuged at 13,000g for 45 minutes at 4°C. The isolated nuclei were then resuspended in Nuclei Wash Buffer at a final concentration of 1,000 nuclei/μl and passed through a 40-μm FlowMi Cell Strainer for further purification. Isolated human nuclei were processed using the Chromium Single Cell 5′ Reagent Kits (10x Genomics) for droplet-based 5′ end massively parallel single-cell RNA sequencing. Sequencing libraries were generated and run on Illumina sequencers at the McDonnell Genome Institute.