Transcriptome-Wide Mapping of m⁶ A and m⁶ Am at Single-Nucleotide Resolution Using miCLIP

The most prevalent modified base in mRNA, N⁶ -methyladenosine (m⁶ A), is found in several thousand transcripts, typically near the stop codon, although it can occur anywhere in the mRNA. In addition, the highly similar nucleotide N⁶ ,2'-O-dimethyladenosine (m⁶ Am), which is difficult to distinguish from m⁶ A, occurs as the first transcribed nucleotide of certain transcripts. Both the m⁶ A and m⁶ Am modifications have been implicated in numerous biological processes, and their precise mapping is crucial to understanding their functions. To address this need, we developed miCLIP, a method that maps both m⁶ A and m⁶ Am at individual nucleotide resolution. Here we describe the miCLIP protocol, with slight improvements to the initially published protocol for both the experimental methodology and bioinformatics analysis. © 2019 by John Wiley & Sons, Inc.