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N 1 -methyladenosine (m 1 A), N 3 -methylcytidine (m 3 C), and N 1 -methylguanosine (m 1 G) 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 m 1 A, m 3 C, or m 1 G using the Escherichia coli dealkylating enzyme AlkB, along with instructions for subsequent processing with widely used protocols for small RNA sequencing.

High-Throughput Small RNA Sequencing Enhanced by AlkB-Facilitated RNA de-Methylation (ARM-Seq)