2.8. m6A MeRIP-Seq (Methylated RNA Immunoprecipitation and Sequencing)

The m⁶A MeRIP-Seq and data analyses were performed as previously described [16]. Briefly, Trizol reagent was used for the isolation of total RNA from muscle cells, and then, the PolyATtract mRNA Isolation System IV (Promega Z5310) was used to enrich mRNA from total RNA sample. 2 μg of the purified mRNA was fragmentized with ZnCl² buffer at 94°C for 5 min. The mRNA fragments were purified and then subjected to immunoprecipitation with anti-m⁶A antibody (1 : 100, Synaptic Systems, Cat No: 202003). After extensive wash, the methylated fragments were eluted by competition using free N6-Methyladenosine (Santa Cruz Biotechnology, sc-215524) and then used for library construction with the TruSeq Stranded mRNA Sample Prep Kits (Illumina RS-122-2101). The input and m⁶A MeRIP libraries were sequenced with Illumina NextSeq 500 with high output kit. The sequence reads mapping, peak calling and visualization, metagene analysis of m⁶A distribution, motif search, and gene ontology analysis were performed as previously described [17].