DART-seq: an antibody-free method for global m ⁶A detection

Abstract

m ⁶A is a widespread RNA modification which influences nearly every aspect of the mRNA life cycle. Our understanding of m ⁶A has been facilitated by the development of global m ⁶A mapping methods, which use antibodies to immunoprecipitate methylated RNA. However, these methods have several limitations, including high input RNA requirements and cross-reactivity to other RNA modifications. Here, we present DART-Seq ( deamination adjacent to RNA modification targets), an antibody-free method for detecting m ⁶A sites. In DART-Seq, the cytidine deaminase APOBEC1 is fused to the m ⁶A-binding YTH domain. APOBEC1-YTH expression in cells induces C to U deamination at sites adjacent to m ⁶A residues, which are detected using standard RNA-Seq. DART-Seq identifies thousands of m ⁶A sites in cells from as little as 10 nanograms of total RNA and can detect m ⁶A accumulation in cells over time. Additionally, we use long-read DART-Seq to gain new insights into m ⁶A distribution along the length of individual transcripts.