27
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: not found

      Transcriptome-wide mapping of N(6)-methyladenosine by m(6)A-seq based on immunocapturing and massively parallel sequencing.

      Nature protocols

      Adenosine, analogs & derivatives, chemistry, Animals, Gene Expression Profiling, methods, Humans, Immunoprecipitation, Methylation, Mice, RNA Processing, Post-Transcriptional

      Read this article at

      ScienceOpenPublisherPubMed
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          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).

          Related collections

          Author and article information

          Journal
          23288318
          10.1038/nprot.2012.148

          Comments

          Comment on this article