Detecting m⁶A with In Vitro DART-Seq

Abstract

Recent studies have uncovered that cellular mRNAs contain a diverse epitranscriptome comprising chemically modified bases which play important roles in gene expression regulation. Among these is m⁶A, which is a highly prevalent modification that contributes to several aspects of RNA regulation and cellular function. Traditional methods for m⁶A profiling have used m⁶A antibodies to immunoprecipitate methylated RNAs. Although powerful, such methods require high amounts of input material. Recently, we developed DART-seq, an antibody-free method for m⁶A profiling from low-input RNA samples. DART-seq relies on deamination of cytidines that invariably follow m⁶A sites and can be performed using a simple in vitro assay with only 50 ng of total RNA. Here, we describe the in vitro DART method and present a detailed protocol for highly sensitive m⁶A profiling from any RNA sample of interest.