GSM8031491: snATACseq_WholeHeart_P8MI_rep2; Mus musculus; ATAC-seq
Atria and aorta were removed from the heart, and the remaining tissue was used for single cell digestion. Cells were harvested at either P5 or P12, from pools of 5 hearts subjected to MI at P1 or P8, respectively. Tissues were minced into small pieces in digestion buffer (HBSS, 1 mg/mL collagenase A) and incubated at 37ºC with agitation for 30 minutes. The suspension was triturated with a 5 mL pipette every 10 minutes. Proper digestion was verified by appearance of single cells under a microscope. The digestion was then diluted 1:1 with HBSS, and the resulting cell suspension was centrifuged at 300 rpm for 6 minutes to remove cardiomyocytes. Supernatant was passed through a 40-micron filter to remove tissue fragments and cardiomyocytes, and then centrifuged at 500 x g for 6 minutes. The cell pellet was resuspended in 1 mL of MACs buffer (PBS, 0.5 % BSA, 2 mM EDTA). The resuspended cells were centrifuged at 500 x g for 6 minutes to remove residual buffer and resuspended in a final volume of 80 uL for incubation with 20 uL of CD31 MACs beads (Miltenyi Biotech). MACs enrichment for CD31+ cells was performed according to the Miltenyi enrichment protocol. Live cells were quantified with Trypan Blue and inCyto C-Chip DHC-F01. For single-nuclei ATAC sequencing, nuclei were isolated from cells according to the 10x protocol. Live nuclei were quantified with DAPI and inCyto C-Chip DHC-F01. scATAC-seq libraries were generated using the 10x Chromium Single Cell 3' Library Kit v3 (10x Genomics) according to manufacturer's instructions. We diluted cell suspensions for a target of 10,000 cells for each library.