RNA modifications play essential roles in gene expression regulation. Only seven out of >150 known RNA modifications are detectable transcriptome-wide by deep sequencing. Here we describe a new principle of RNAseq library preparation, which relies on a chemistry based positive enrichment of reads in the resulting libraries, and therefore leads to unprecedented signal-to-noise ratios. The proposed approach eschews conventional RNA sequencing chemistry and rather exploits the generation of abasic sites and subsequent aniline cleavage. The newly generated 5'-phosphates are used as unique entry for ligation of an adapter in library preparation. This positive selection, embodied in the AlkAniline-Seq, enables a deep sequencing-based technology for the simultaneous detection of 7-methylguanosine (m⁷ G) and 3-methylcytidine (m³ C) in RNA at single nucleotide resolution. As a proof-of-concept, we used AlkAniline-Seq to comprehensively validate known m⁷ G and m³ C sites in bacterial, yeast, and human cytoplasmic and mitochondrial tRNAs and rRNAs, as well as for identifying previously unmapped positions.

• PMID: 30370969
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Marchand V, Ayadi L, Ernst FGM, Hertler J, Bourguignon-Igel V, Galvanin A, Kotter A, Helm M, Lafontaine DLJ, Motorin Y

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