M6a nucleotide solution

The MeRIP assay was performed as reported in our previous study (33 (link)). Briefly, anti-m6A primary antibody (Synaptic Systems) was incubated with Pierce™ Protein A/G Magnetic Beads (Thermo Scientific) at 4°C for 3 h. Then, RNA was fragmented with an RNA fragmentation kit (Ambion) and then incubated with the mixture of Protein A/G and m6A-antibody at 4°C overnight. The immunoprecipitated RNA was washed five times and eluted from beads with m6A nucleotide solution (Sigma-Aldrich) and then purified for RT-qPCR assays.

MeRIP-seq and data analysis were performed as described previously [46 (link)]. Briefly, total RNA was extracted from cells using Trizol (Invitrogen) following the manufacturer’s instructions. mRNA was first purified from total RNA using Gen Elute mRNA Miniprep Kit (Sigma-Aldrich). Then 5 μg mRNA was fragmented using a RNA fragmentation kit (Ambion) and immunoprecipitated with the mixture of Protein A beads (Thermo Fisher) and anti-m⁶A antibody (Synaptic Systems); the immunoprecipitated RNA was washed and eluted by competition with m⁶A nucleotide solution (Sigma-Aldrich). The purified RNA fragments from m⁶A MeRIP were used for library construction and sequenced with Illumina Nextseq500. Reads mapping, m⁶A peak calling, motif search, and differentially methylated peaks were analyzed by exomePeak R/Bioconductor package as described [47 (link)].