High-Throughput Small RNA Sequencing Enhanced by AlkB-Facilitated RNA de-Methylation (ARM-Seq)
N1-methyladenosine (m1A), N3-methylcytidine (m3C), and N1-methylguanosine (m1G) are common in transfer RNA (tRNA) and tRNA-derived fragments. These modifications alter Watson-Crick base-pairing, and cause pauses or stops during reverse transcription required for most high-throughput RNA sequencing protocols, resulting in inefficient detection of methyl-modified RNAs. Here, we describe a procedure to demethylate RNAs containing m1A, m3C, or m1G using the Escherichia coli dealkylating enzyme AlkB, along with instructions for subsequent processing with widely used protocols for small RNA sequencing.
Key wordsRNA Sequencing Transfer RNA (tRNA) AlkB RNA demethylation N1-methyladenosine (m1A) N3-methylcytidine (m3C) N1-methylguanosine (m1G)
This work was supported by the US National Institutes of Health (NIH) NHGRI grant 5R01HG006753 to T.M.L. and by NIH grant GM052347 to E.M.P. Thanks are given to Patricia Chan for the assistance in the preparation of the figures.
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