Abstract
N(6)-methyladenosine-sequencing (m(6)A-seq) is an immunocapturing approach for the unbiased transcriptome-wide localization of m(6)A in high resolution. To our knowledge, this is the first protocol to allow a global view of this ubiquitous RNA modification, and it is based on antibody-mediated enrichment of methylated RNA fragments followed by massively parallel sequencing. Building on principles of chromatin immunoprecipitation-sequencing (ChIP-seq) and methylated DNA immunoprecipitation (MeDIP), read densities of immunoprecipitated RNA relative to untreated input control are used to identify methylated sites. A consensus motif is deduced, and its distance to the point of maximal enrichment is assessed; these measures further corroborate the success of the protocol. Identified locations are intersected in turn with gene architecture to draw conclusions regarding the distribution of m(6)A between and within gene transcripts. When applied to human and mouse transcriptomes, m(6)A-seq generated comprehensive methylation profiles revealing, for the first time, tenets governing the nonrandom distribution of m(6)A. The protocol can be completed within ~9 d for four different sample pairs (each consists of an immunoprecipitation and corresponding input).