GSM7231237: SC_39_3 WT(E410) with SHAM Unx + VEH; Mus musculus; ATAC-seq
A 2 mm3 section of tissue collected from perfused cortex stored at -80C was rinsed briefly with PBS and minced before adding 1 mL of lysis buffer containing 20 mM Tris-HCl (pH 8), 320 mM sucrose, 5 mM CaCl2, 3 mM MgAc2, 0.1 mM EDTA, 0.1% TritonX-100, 0.1% RNAse Inhibitor and 0.1% DAPI. The tissue was initially dissociated by pipetting up and down 10X with a p-1000 tip and then being passed through a 25G syringe 10X. The tissue was incubated on ice for 10 minutes and then passed through a 30 µm CellTrics filter. Nuclei were pelleted by centrifugation (5 min, 500g, 4°C) and washed with PBS after removal of supernatant. Nuclei were pelleted again and resuspended in 1 mL of PBS containing 0.04% BSA and RNAse inhibitor (0.2u/µL) and then passed through a 40 µm FlowMI filter before generating counts with hemocytometer. Nuclei were then diluted and prepared for snATA-seq with the 10X Chromium System according to the manufacturer's instructions (10X Genomics) ATAC libraries were prepared for sequencing using standard 10X Genomics and Illumina protocols