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Transcriptome-wide mapping of N 6-methyladenosine by m6A-seq based on immunocapturing and massively parallel sequencing

Transcriptome-wide mapping of N 6-methyladenosine by m6A-seq based on immunocapturing and... N 6-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 m6A 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). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Protocols Springer Journals

Transcriptome-wide mapping of N 6-methyladenosine by m6A-seq based on immunocapturing and massively parallel sequencing

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References (44)

Publisher
Springer Journals
Copyright
Copyright © 2013 by Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.
Subject
Life Sciences; Life Sciences, general; Biological Techniques; Analytical Chemistry; Microarrays; Computational Biology/Bioinformatics; Organic Chemistry
ISSN
1754-2189
eISSN
1750-2799
DOI
10.1038/nprot.2012.148
Publisher site
See Article on Publisher Site

Abstract

N 6-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 m6A 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).

Journal

Nature Protocols – Springer Journals

Published: Jan 3, 2013

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