An experimental workflow for identifying RNA m 6 A alterations in cellular senescence by methylated RNA immunoprecipitation sequencing.

N-methyladenosine (mA), the most prevalent mRNA modification in eukaryotic cells, is known to play regulatory roles in a wide array of biological processes, including aging and cellular senescence. To investigate such roles, the mA modification can be identified across the entire transcriptome by immunoprecipitation of methylated RNA with an anti-mA antibody, followed by high-throughput sequencing (meRIP-seq or mA-seq). Presented here is a protocol for employing meRIP-seq to profile the RNA mA landscape in senescent human cells. We described, in detail, sample preparation, mRNA isolation, immunoprecipitation, library preparation, sequencing, bioinformatic analysis and validation. We also provided tips and considerations for the optimization and interpretation of the results. Our protocol serves as a methodological resource for investigating transcriptomic mA alterations in cellular senescence as well as a valuable paradigm for the validation of genes of interest.