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Transcriptome-wide m6A detection with DART-Seq

Publication ,  Journal Article
Meyer, KD
September 23, 2019

mA is the most abundant internal mRNA modification and plays diverse roles in gene expression regulation. Much of our current knowledge about mA has been driven by recent advances in the ability to detect this mark transcriptome-wide. Antibody-based approaches have been the method of choice for global mA mapping studies. These methods rely on mA antibodies to immunoprecipitate methylated RNAs, followed by next-generation sequencing to identify mA-containing transcripts. While these methods enabled the first identification of mA sites transcriptome-wide and have dramatically improved our ability to study mA, they suffer from several limitations. These include requirements for high amounts of input RNA, costly and time-consuming library preparation, high variability across studies, and mA antibody cross-reactivity with other modifications. Here, we describe DART-Seq (eamination djacent to NA modification argets), an antibody-free method for global mA detection. In DART-Seq, the C to U deaminating enzyme, APOBEC1, is fused to the mA-binding YTH domain. This fusion protein is then introduced to cellular RNA either through overexpression in cells or with assays, and subsequent deamination of mA-adjacent cytidines is then detected by RNA sequencing to identify mA sites. DART-Seq can successfully map mA sites throughout the transcriptome using as little as 10 nanograms of total cellular RNA, and it is compatible with any standard RNA-seq library preparation method.

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Publication Date

September 23, 2019
 

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Meyer, K. D. (2019). Transcriptome-wide m6A detection with DART-Seq. https://doi.org/10.21203/rs.2.12189/v1
Meyer, Kate D. “Transcriptome-wide m6A detection with DART-Seq,” September 23, 2019. https://doi.org/10.21203/rs.2.12189/v1.
Meyer, Kate D. Transcriptome-wide m6A detection with DART-Seq. Sept. 2019. Crossref, doi:10.21203/rs.2.12189/v1.

DOI

Publication Date

September 23, 2019