GSM2525591: atac_P1_rep1; Mus musculus; ATAC-seq
Mouse hearts were pooled to obtain enough material for sequencing (P1: 12 hearts/sample, P14 and P56: 6-7 hearts/sample). The samples were homogenized in 15 mL lysis buffer (0.32 M sucrose, 10 mM Tris-HCl (pH = 8), 5 mM CaCl2, 5 mM magnesium acetate, 2 mM EDTA, 0.5 mM EGTA, 1 mM DTT and 1X complete protease inhibitor (all chemical reagents were acquired from Sigma-Aldrich). The heart lysate was combined with another 15 mL lysis buffer and subsequently homogenized for 10-15 strokes using a dounce tissue grinder (Wheaton, Millville, NJ, USA). The cell lysate was sequentially filtered through 70 μM and 40 μM cell strainers (Becton Dickinson, Franklin Lakes, NJ, USA) and centrifuged to pellet nuclei at 1000 x g for 5 mins. Nuclei pellets were then resuspended in 2-4mL sucrose buffer (1 M sucrose, 10 mM Tris-HCl (pH = 8), 5 mM magnesium acetate, 1 mM DTT and 1X complete protease inhibitor) and the suspension was cushioned on top of a 2x volume of the sucrose buffer, followed by centrifugation to pellet nuclei at 1000 xg for 5 mins. Nuclei pellets were then washed once in 1 mL of nuclei storage buffer (NSB, 0.44 M sucrose, 10 mM Tris-HCl (pH = 7.2), 70 mM KCl, 10 mM MgCl2, 1.5 mM spermine and 1x complete protease inhibitor). The washed nuclei pellets were then resuspended in NSB and stained for the cardiomyocyte nuclear specific marker, Pcm1 (1:200, HPA023374, Sigma-Aldrich) at 4oC for 45 minutes. Omitting the primary antibody served as a control for positive staining. Nuclei were washed twice in NSB and stained with secondary antibody conjugated with Alexa Fluor 633 (1:500, ThermoFisher Scientific (Molecular Probes)) or Brilliant Violet 421 (1:400, BioLegend, San Diego, CA, USA) at 4oC for 30 minutes. Nuclei were washed twice in NSB and resuspended in PBS before being processed for FACS. Pcm1 (-/+) nuclei were separated using the BD FACS ARIA cell sorter. Collected Pcm1 (-/+) nuclei were pelleted at 1500 x g for 15 minutes. The purity of nuclei isolations was checked by qPCR of cardiomyocyte enriched genes. ATAC-seq library preparation. Nuclei pellets underwent the transposition reaction using Nextera DNA library preparation kit (FC-121-1030, Illumina) followed by purification using MinElute PCR purification kit (#28004, Qiagen) and PCR amplification. Size selection by agarose gel was performed after PCR to select for library fragment sizes of 200-400bp. Libraries were validated using the MultiNA bioanalyzer (Shimadzu). Libraries were sequenced with the HiSeq2500 using version 4 reagents (Illumina), yielding 100 bp single end reads. Base-calling was performed with RTA (version 1.8.66.3) and converted to fastq file using bcl2fastq (version 2.17.1.14).