GSM5470488: s15; Homo sapiens; RNA-Seq
Snap-frozen autopsy human brain tissue sampling used a 3mm-punch biopsy tool and a cold blade. Nuclei extraction from snap-frozen human autopsy brains was performed according to the following protocol. On wet ice, the frozen, dissected tissue was lysed using a Dounce homogenizer (10 strokes with the loose pestle followed by 20 strokes with the tight pestle) in 600-μl of freshly made cold lysis buffer (low sucrose buffer, Triton X-100 20%, Rnase inhibitor 40U/μl). Part of the homogenate (200 μl) was collected for bulk RNA extraction (Qiagen RNAeasy kit) and stored at -80°C for future applications. The remaining homogenate was filtered through a 40-μm cell strainer for nuclei extraction with 4-ml of low sucrose buffer (0.64M sucrose, HEPES pH8, CaCl2, MgAc, EDTA, DTT, nuclease-free water). Sonication of the homogenate was performed for 10 sec. To remove cellular and myelin debris, a sucrose gradient (12-ml of high sucrose buffer [2M sucrose, HEPES pH8, MgAc, DTT, nuclease-free water] underneath the low sucrose buffer) was centrifuged at 3,200 g for 30 minutes, and the supernatant was discarded. Nuclei were resuspended in 1-ml resuspension solution (PBS, 5% BSA and RNAse inhibitor 40U/μl) and centrifuged for 12-min at 3,200 g. Nuclei were again resuspended in 50-μl cold resuspension solution. Nuclei were counted using trypan blue and a haemocytometer under bright field. A yield of 700–1,200 nuclei/μl per sample was targeted. Immediately after extraction, 5,000 nuclei/sample were loaded onto a Chromium Single Cell 3′ Chip (10X Genomics) and processed for the single-nucleus cDNA library preparation (Chromium Single Cell 3' version 3, 10X Genomics) according to the manufacturer's instructions without modifications. Chromium Single Cell 3' version 3, 10X Genomics