Document Detail

Transcriptome-wide mapping of N(6)-methyladenosine by m(6)A-seq based on immunocapturing and massively parallel sequencing.
MedLine Citation:
PMID:  23288318     Owner:  NLM     Status:  In-Data-Review    
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).
Dan Dominissini; Sharon Moshitch-Moshkovitz; Mali Salmon-Divon; Ninette Amariglio; Gideon Rechavi
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Publication Detail:
Type:  Journal Article     Date:  2013-01-03
Journal Detail:
Title:  Nature protocols     Volume:  8     ISSN:  1750-2799     ISO Abbreviation:  Nat Protoc     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-01-04     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101284307     Medline TA:  Nat Protoc     Country:  England    
Other Details:
Languages:  eng     Pagination:  176-89     Citation Subset:  IM    
1] Cancer Research Center, Chaim Sheba Medical Center, Tel Hashomer, Israel. [2] Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel. [3].
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