Transcriptome-wide mapping of N⁶-methyladenosine by m ⁶A-seq based on immunocapturing and massively parallel sequencing

N⁶-methyladenosine-sequencing (m⁶A-seq) is an immunocapturing approach for the unbiased transcriptome-wide localization of m⁶A 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 ⁶A between and within gene transcripts. When applied to human and mouse transcriptomes, m⁶A-seq generated comprehensive methylation profiles revealing, for the first time, tenets governing the nonrandom distribution of m⁶A. The protocol can be completed within ~9 d for four different sample pairs (each consists of an immunoprecipitation and corresponding input).