GSM6165755: P33, CD14, atac; Homo sapiens; ATAC-seq
Nuclear isolation, library preparation, and quality control were performed according to Hill et. al. (2021) for PCM1 isolation and ATAC-seq. Briefly, fresh cardiac tissue was harvested on ice and immediately homogenized with a Biogen Series PRO200 (PRO Scientific) prior to dounce homogenization. Nuclei were isolated via density gradient centrifugation with optiprep density gradient medium (Sigma). All nuclei isolated from 30-40% interface were pre-cleared with Protein-G Dynabeads (Life technologies). Next, nuclei were immunoprecipitated with an anti-PCM1 (Sigma, HPA023370) antibody and Protein-G Dynabeads to enrich for cardiomyocyte nuclei. CD14+ cells were isolated by using the CD14 MicroBeads (Miltenyi Biotec, 130-050-201) as per the manufacturer's instructions. Briefly, PBMCs were washed twice with Dulbecco's PBS containing 2% FBS (STEMCELL technologies, #07905) before being bound to CD14 MicroBeads and isolated with MS columns (Miltenyi Biotec, 130-042-201). Cells were subjected to two rounds of purification before ATAC-seq. NK cells were isolated from CD14+ cell–depleted PBMC pools by taking the flow through from the CD14+ MACS enrichment. After flowing through the MACS MS columns, NK cells were isolated by using the human NK cell isolation kit (Miltenyi Biotec, 130-092-657) according to the manufacturer's instructions, allowing for the isolation of both cell types simultaneously from the same patient sample. After the purification of NK cells and CD14+ monocytes from density gradient–purified PBMCs, we performed ATAC-seq by using the Omni-ATAC protocol with roughly 50,000 cells as input. OMNI-ATAC protocol with 50,000 cells or cardiomyocyte nuclei as input.