Detecting m 6 A with In Vitro DART-Seq

Methods Mol Biol. 2022;2404:363-374. doi: 10.1007/978-1-0716-1851-6_20.

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.

Keywords: DART-seq; Epitranscriptome; In vitro deamination; Protein purification; RNA isolation; m6A.