GSM7062662: Male CCI high biol rep 2, Day3; Mus musculus; RNA-Seq
For single nuclei isolation, mice were transcardially perfused with 0.1M PBS and brains were dissected on ice. A 3mm perilesional coronal section was isolated from the ipsilateral hemisphere (AP Bregma -1 mm to -4 mm), and a block containing hippocampal and cortical tissue was then further dissected from that section (DV Bregma 0 mm to -3 mm).  The dissected tissue block was rapidly frozen and kept at -80°C before nuclear isolation.  Tissue samples were homogenized in homogenization buffer (0.32M sucrose, 5mM MgAc2, 10mM Tris-HCl pH 8.0, 0.1% Triton-X-100, 0.1mM EDTA with PierceTM protease inhibitors). The resulting homogenate was layered onto a sucrose cushion (1.8M sucrose, 10mM Tris-HCl pH 8.0, 3mM MgAc2, with PierceTM protease inhibitors). Tubes were loaded into an SW 32 Ti swinging bucket and centrifuged in a Beckman Coulter Optima X Series ultracentrifuge at 25,000 rpm at 4°C for 2 hr. Nuclei were treated with a nuclease inhibitor and resuspended in 0.04% BSA in DPBS. Isolated nuclei were processed using the 10x Genomics GEM Single Cells 3' Kit v3.1 reagents per manufacturer's instructions to generate barcoded single cell RNAseq libraries.  Briefly, an aliquot of each sample was diluted 1:20 with trypan blue and counted on a hemocytometer.  Based on the nuclei count, each sample was diluted to a concentration of 1200 nuclei/ml using PBS+ 0.04% BSA + 0.2 units/ml RNAse inhibitor (Millipore Sigma). The viability of the samples was 91 – 96% prior to loading the samples on a Chip G. Approximately 12,000 cells/samples were loaded on the chip with a target cell recovery of 10,000/sample, and the samples were visually observed to be opaque and uniform. Individual cell's RNA was barcoded then stored at -20°C for < 1 week until RNAseq libraries were made.  The libraries were built using reagents provided in the 10X Genomics kit with 10X Genomics UDI's as per manufacturer's directions. The quality of the libraries was determined by assaying an aliquot on a high sensitivity D1000 screen tape.