An experimental workflow for identifying RNA m⁶A alterations in cellular senescence by methylated RNA...

N⁶-methyladenosine (m⁶A), 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 m⁶A modification can be identified across the entire transcriptome by immunoprecipitation of methylated RNA with an anti-m⁶A antibody, followed by high-throughput sequencing (meRIP-seq or m⁶A-seq). Presented here is a protocol for employing meRIP-seq to profile the RNA m⁶A 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 m⁶A alterations in cellular senescence as well as a valuable paradigm for the validation of genes of interest.