Abstract
Epitranscriptomics is an emerging field where the development of high-throughput analytical technologies is essential to profile the dynamics of RNA modifications under different conditions. Despite important advances during the last 10years, the number of RNA modifications detectable by next-generation sequencing is restricted to a very limited subset. Here, we describe a highly efficient and fast method called AlkAniline-Seq to map simultaneously two different RNA modifications: 7-methyl-guanosine (m7G) and 3-methyl-cytosine (m3C) in RNA. Our protocol is based on three subsequent chemical/enzymatic steps allowing the enrichment of RNA fragments ending at position n+1 to the modified nucleotide, without any prior RNA selection. Therefore, AlkAniline-Seq demonstrates an outstanding sensitivity and specificity for these two RNA modifications. We have validated AlkAniline-Seq using bacterial, yeast, and human total RNA, and here we present, as an example, a synthetic view of the complete profiling of these RNA modifications in S. cerevisiae tRNAs.
Published Version
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