GSM8527854: ventricle, 24 month,  scATAC, sample 2; Mus musculus; ATAC-seq
Male mouse hearts at 3 months of age, 12 months of age, and 24 months of age were collected for non-cardiomyocyte isolation. In accordance with UNC-Chapel Hill LARC guidelines, all animals were euthanized using CO2 and cervical dislocation. Hearts were perfused with Langendorff apparatus as described with minor modifications. Briefly, male mice of three ages groups were anesthetized with isoflurane (1%-2%), the heart was removed, retrograde perfusion was performed through an aortic cannulation, and continuous flow of 16-24 drops/min in a Langendorff apparatus. Both ventricles were digested with 1 mg/ml collagenase IaI for 10-15min, and the ventricle were further mechanically dissociated by pipetting. Then, the cell suspension was collected and filtered through a 100µm cell strainer. CMs were removed by centrifuging at 50g (200 rpm) for 3min, and nonCMs were enriched by centrifuge at 450g (2,000 rpm) for 5 min. The pelleted nonCMs were treated with 1ml red blood cell lysis buffer (Gibco) at room temperature (RT) and washed twice with wash buffer (4% FBS/PBS). Single live cells were FACS-sorted by 7-Amino Actinomycin D (7-AAD, R&D Systems), and Pacific Blue-conjugated Annexin V and SYTOX (Thermo) staining. Following sorting, cell concentration and viability of each sample were counted using automated cell counter (LUNA system, Logos biosytems) plus Acridine orange (AO)/ propidium iodide (PI) dual nuclear staining. Cell concentration was adjusted to 1x106 cells/ml for subsequent scRNA-seq via 10x Gemonics platform. Cells (50,000 per sample) for scATAC-seq were subjected to lysis for 3min and counted again with cell counter to determine the viability (lower than 5%) and concentration again. Single-cell ATAC library preparation and sequencing: The single nuclei were processed for library preparation (Chromium Single Cell ATAC v1.0) following manufactural instructions from 10x Chromium. Sequencing was performed on Illumina NOVAseq-S1 platform.