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

Dan Dominissini, Sharon Moshitch-Moshkovitz, Mali Salmon-Divon, Ninette Amariglio, Gideon Rechavi

Research output: Contribution to journalArticlepeer-review

473 Scopus citations

Abstract

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).

Original languageEnglish
Pages (from-to)176-189
Number of pages14
JournalNature Protocols
Volume8
Issue number1
DOIs
StatePublished - Jan 2013
Externally publishedYes

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