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

  • Anya V. Grozhik
  • Bastian Linder
  • Anthony O. Olarerin-George
  • Samie R. Jaffrey
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1562)

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.

Key words

RNA N6-Methyladenosine Crosslinking High-throughput sequencing 

Notes

Acknowledgments

We thank members of the Jaffrey lab for their helpful comments and support. This work was supported by NIH grants NIDA DA037150 (S.R.J.), T32 CA062948 (A.O.-G.), and a German Research Foundation (DFG) fellowship (B.L.).

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Anya V. Grozhik
    • 1
  • Bastian Linder
    • 1
  • Anthony O. Olarerin-George
    • 1
  • Samie R. Jaffrey
    • 1
  1. 1.Department of Pharmacology, Weill Medical CollegeCornell UniversityNew YorkUSA

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