title: High-Throughput Small RNA Sequencing Enhanced by AlkB-Facilitated RNA de-Methylation (ARM-Seq).
abstract: 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.
authors.affiliation: Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA, USA.
authors.forename: Eva
authors.initials: E
authors.lastname: Hrabeta-Robinson
authors.affiliation: Department of Biochemistry & Biophysics, Center for RNA Biology, University of Rochester School of Medicine, Rochester, NY, 14642, USA.
authors.forename: Erin
authors.initials: E
authors.lastname: Marcus
authors.affiliation: Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA, USA.
authors.forename: Aaron E
authors.initials: AE
authors.lastname: Cozen
authors.affiliation: Department of Biochemistry & Biophysics, Center for RNA Biology, University of Rochester School of Medicine, Rochester, NY, 14642, USA.
authors.forename: Eric M
authors.initials: EM
authors.lastname: Phizicky
authors.affiliation: Department of Biomolecular Engineering, University of California Santa Cruz, Physical Sciences Bldg., Room 320, 1156 High Street, Santa Cruz, CA, 95064, USA. tmjlowe@ucsc.edu.
authors.forename: Todd M
authors.initials: TM
authors.lastname: Lowe
mesh_terms: D000071496:AlkB Enzymes / Q000378:metabolism*; D000818:Animals; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008745:Methylation; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA
chemical_list: D012313:RNA; D012343:RNA, Transfer; D000071496:AlkB Enzymes
keywords: AlkB; N 1-methyladenosine (m1A); N 1-methylguanosine (m1G); N 3-methylcytidine (m3C); RNA Sequencing; RNA demethylation; Transfer RNA (tRNA)