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Methods Mol Biol. 2017;1562:55-78. doi: 10.1007/978-1-4939-6807-7_5.

Mapping m6A at Individual-Nucleotide Resolution Using Crosslinking and Immunoprecipitation (miCLIP).

Author information

1
Department of Pharmacology, Weill Medical College, Cornell University, New York, NY, 10065, USA.

Abstract

N 6 -methyladenosine (m6A) is the most abundant modified base in eukaryotic mRNA and has been linked to diverse effects on mRNA fate. Current m6A mapping approaches localize m6A residues to 100-200 nt-long regions of transcripts. The precise position of m6A in mRNAs cannot be identified on a transcriptome-wide level because there are no chemical methods to distinguish between m6A and adenosine. Here, we describe a method for using anti-m6A antibodies to induce specific mutational signatures at m6A residues after ultraviolet light-induced antibody-RNA crosslinking and reverse transcription. Then, we describe how to use these mutational signatures to map m6A residues at nucleotide resolution. Taken together, our protocol allows for high-throughput detection of individual m6A residues throughout the transcriptome.

KEYWORDS:

Crosslinking; High-throughput sequencing; N 6 -Methyladenosine; RNA

PMID:
28349454
PMCID:
PMC5562447
DOI:
10.1007/978-1-4939-6807-7_5
[Indexed for MEDLINE]
Free PMC Article

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