Transcriptome-Wide Mapping of m 6 A and m 6 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.