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

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

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.

MeSH terms

  • Adenosine / analogs & derivatives*
  • Antibodies
  • Computational Biology / methods
  • Genome
  • High-Throughput Nucleotide Sequencing*
  • Immunoprecipitation* / methods
  • Isotope Labeling
  • Polymerase Chain Reaction
  • RNA / chemistry
  • RNA / genetics*
  • Software

Substances

  • Antibodies
  • RNA
  • N-methyladenosine
  • Adenosine