m6A RNA Immunoprecipitation Followed by High-Throughput Sequencing to Map N6-Methyladenosine.

ABSTRACT

N6-methyladenosine (m6A) is the most abundant internal modification on messenger RNAs (mRNAs) and long noncoding RNAs (lncRNAs) in eukaryotes. It influences gene expression by regulating RNA processing, nuclear export, mRNA decay, and translation. Hence, m6A controls fundamental cellular processes, and dysregulated deposition of m6A has been acknowledged to play a role in a broad range of human diseases, including cancer. m6A RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq or m6A-seq) is a powerful technique to map m6A in a transcriptome-wide level. After immunoprecipitation of fragmented polyadenylated (poly(A)+) rich RNA by using specific anti-m6A antibodies, both the immunoprecipitated RNA fragments together with the input control are subjected to massively parallel sequencing. The generation of such comprehensive methylation profiles of signal enrichment relative to input control is necessary in order to better comprehend the pathogenesis behind aberrant m6A deposition.