GSM5681674: 036 Coronary artery bulk ATAC; Homo sapiens; ATAC-seq
Frozen coronary artery segments were broken down into small pieces using mortar and pestle under dry ice and liquid nitrogen. Nuclei were isolated using iodixanol/sucrose gradients following the Omni-ATAC protocol (Corces et al. Nature Methods 2017). 50,000 nuclei were washed in cold ATAC Resuspension Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) + 0.1% Tween-20 and centrifuged for 10 minutes at 500 g at 4°C. 50,000 nuclei were then resuspended in 50 µl of transposition mix (25 µl 2X TD buffer, 2.5 µl of Tn5 transposase (Illumina), 16.5 µl PBS, 0.01% digitonon, 0.1% Tween-20). Samples were subsequently transposed for 30 minutes at 37°C with shaking (1000 rpm). Transposed DNA was first purified using the Zymo DNA Clean and Concentrator-5 Kit. Libraries were subsequently constructed and amplified according to Supplementary Protocol 1 in the Omni-ATAC protocol (Corces et al. Nature Methods 2017). Each library underwent 5 cycles of pre-amplification and then additional amplification cycles (determined by qPCR of pre-amplified mixtures)