Detecting mA with In Vitro DART-Seq.

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 mA, which is a highly prevalent modification that contributes to several aspects of RNA regulation and cellular function. Traditional methods for mA profiling have used mA 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 mA profiling from low-input RNA samples. DART-seq relies on deamination of cytidines that invariably follow mA 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 mA profiling from any RNA sample of interest.