Mapping single-nucleotide m6A by m6A-REF-seq

Abstract

The past few years have witnessed rapid progress in the field of RNA modifications. As the most prevailing modification on eukaryotic mRNA, m⁶A is characterized to play a vital role in various cellular activities. However, limitations of the detection method impede functional studies of m⁶A. Here we introduce m⁶A-REF-seq, a powerful and straightforward method to identify m⁶A at single-nucleotide resolution. m⁶A-REF-seq relies on the recognition of RNA endonuclease MazF towards m⁶A at the ACA motif, providing an orthogonal method independent of the m⁶A antibody being adopted by most of current methods. We describe a detailed protocol to perform m⁶A-REF-seq, including NGS library construction and sequencing data analysis. In particular, we describe an optimized assay to validate individual m⁶A sites identified by m⁶A-REF-seq, which can also be applied to detect any candidate m⁶A sites.

Keywords: Epitranscriptome; RNA modification; Single-base resolution; m(6)A.