Improved methods for deamination-based m6A detection

We present an improved version of DART-seq which utilizes a variant of the YTH domain engineered to achieve enhanced m6A recognition in APO1-YTH (D422N). In addition, we develop in vitro DART-seq and show that it performs similarly to cellular DART-seq and can map m6A in any sample of interest using nanogram amounts of total RNA. Overall design: Cellular DART-seq: We performed RNA-seq on RNA from HEK293T cells expressing various DART constructs (containing APO domain or YTH domain variants) tested in the paper. We also sequenced RNA from cells expressing APO1-YTHmut, ADAR-YTHmut, or ADAR alone which serves as controls. There are at least 3 biological replicates included for each of these samples. In vitro DART-seq: We performed RNA-seq on RNA columned purified after incubation under various in vitro DART-seq conditions (time, protein:RNA, protein variant). We also sequenced RNA column purified after incubation with APO1-YTHmut or treated under YTH blocking conditions as negative control for in vitro DART-seq.