NOMe-seq library preparation and sequencing

To optimize the NOMe-seq procedure for frozen heart tissue samples, we modified the protocol based on previous studies [29,34,85,86]. Approximately 30 mg tissues were used for NOMe-seq in this study. In brief, the heart tissues were thoroughly ground at low temperatures. The tissue pellets were resuspended in 500 μl ice-cold lysis buffer with Protease Inhibitor Cocktail: 10 mM Tris·HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl₂, 0.1 mM EDTA, and 0.5% NP-40. The tubes were incubated for 60 min with vortexing every 10 min to release nuclei. The nuclei were washed twice with cold DPBS, and the lysates were incubated in 60 units of M.CviPI GpC Methyltransferase (NEB, Cat. M0227L) for 1 h, with the addition of 3 ng unmodified lambda DNA (Thermo Fisher Scientific, Waltham, Massachusetts, Cat. SD0021). Twenty units of M.CviPI and 0.75 μl of 200× SAM were added for another 1 h incubation. The reactions were then stopped by adding EDTA and proteinase K to digest the proteins overnight. Genomic DNAs were purified by phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation. Next, bisulfite conversion of genomic DNAs was conducted using EZ-96 DNA Methylation-Direct MagPrep (Zymo Research, Irvine, California, USA, Cat. D5044). Oligo1 (5′-biotin- CTACACGACGCTCTTCCGATCTNNNNNNNNN-3′) and Oligo2 (AGACGTGTGCTCTTCCGATCTNNNNNNNNN) were used to synthesize the first and second strands, respectively. Finally, the NOMe-seq libraries were amplified with 12 cycles of PCR using KAPA HiFi Hot Start Ready Mix (KAPA Biosystems, Cat. KK2602). The NOMe-seq libraries and RNA-seq libraries were sequenced on the HiSeq 4000 platform (Novogene, Beijing, China).