Transcriptome-Wide Mapping of m6A and m6Am at Single-Nucleotide Resolution Using miCLIP

The most prevalent modified base in mRNA, N⁶-methyladenosine (m⁶A), 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 (m⁶Am), which is difficult to distinguish from m⁶A, occurs as the first transcribed nucleotide of certain transcripts. Both the m⁶A and m⁶Am 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 m⁶A and m⁶Am 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.