AlkB-facilitated RNA Methylation Sequencing (ARM-Seq) of Saccharomyces cerevisiae small RNAs

Study goals for AlkB-facilitated RNA Methylation Sequencing (ARM-Seq) were to identify small RNAs containing certain nucleoside modifications that are known to interfere with reverse transcription required for RNA-seq library preparation. In order to facilitate sequencing RNA samples were treated using the Escherichia coli dealkylating enzyme AlkB to demethylate 1-methyladenosine (m1A), 3-methylcytidine (m3C) and 1-methylguanosine (m1G) residues prior to RNA-seq library preparation. These modifications are known to be common in transfer RNAs (tRNAs). Without treatment RNAs containing m1A, m3C or m1G modifications are expected to block progression of reverse transcriptase, resulting in failure of PCR amplification and sequencing, and under-representation in sequencing results. Modified RNAs revealed by ARM-Seq were identified by comparing sequencing results for AlkB-treated samples to those from untreated samples using software for differential expression analyses.