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Meyer, Kate D
Nature methods, 12/2019, Letnik: 16, Številka: 12Journal Article
N.sup.6-methyladenosine (m.sup.6A) is a widespread RNA modification that influences nearly every aspect of the messenger RNA lifecycle. Our understanding of m.sup.6A has been facilitated by the development of global m.sup.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.sup.6A sites. In DART-seq, the cytidine deaminase APOBEC1 is fused to the m.sup.6A-binding YTH domain. APOBEC1-YTH expression in cells induces C-to-U deamination at sites adjacent to m.sup.6A residues, which are detected using standard RNA-seq. DART-seq identifies thousands of m.sup.6A sites in cells from as little as 10 ng of total RNA and can detect m.sup.6A accumulation in cells over time. Additionally, we use long-read DART-seq to gain insights into m.sup.6A distribution along the length of individual transcripts. Getting around the limitations of antibody-based N.sup.6-methyladenosine (m.sup.6A) pulldown, such as high input requirements and cross-reactivity, DART-seq profiles transcriptome-wide m.sup.6A occurrences from RNA amounts equivalent to the RNA obtained from 1,000 cells.
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