GSM8244646: ATAC temporal neocortex, nonepileptic,replicate2; Homo sapiens; ATAC-seq
Each brain tissue sample was cut into small pieces and then transferred to a Dounce homogenizer with 400 μl of lysis buffer (pH 7.4, 10 mM Tris-HCL, 3 mM MgCl2, 10 mM NaCl, 0.1% IGEPAL, 0.1% Tween 20, 2% BSA). To release the nuclei, the tissues were homogenized. After 10 minutes of centrifugation at 4°C, the nuclei were washed in 1 ml of cold nuclei wash and resuspension buffer (1× PBS supplemented with 0.2 U/μl RNase inhibitor and 1% BSA). Trypan blue and Countess II FL Automated Cell Counter/Microscopy were used to evaluate lysis efficiency and viability. A 40 μm filter was used for the elimination of large clumps and cell debris. Another centrifugation step was performed, and the nuclei were collected for direct myelin removal. Then, 500 μl or an appropriate volume of nucleus wash and resuspension buffer was added to the nucleus pellet to reach the desired 700-1,200 nuclei/μl concentration. We loaded the nucleus suspension into the Chromium Next GEM Chip H with 10x reagents and labelled it with barcodes using a 10×Chromium Controller (10X Genomics). Following the amplification of DNA fragments from the cells labelled with barcodes, sequencing libraries were prepared using reagents from the Chromium NextGEM Single Cell ATAC Reagent Kit v1.1 (10x Genomics) following the manufacturer's guidelines. The scATAC-seq libraries were constructed following the Chromium NextGEM Single Cell ATAC Reagent Kit v1.1 User Guide RevC (10x Genomics CG000209).