title: Biochemical and Transcriptome-Wide Identification of A-to-I RNA Editing Sites by ICE-Seq.
abstract: Inosine (I) is a modified adenosine (A) in RNA. In Metazoa, I is generated by hydrolytic deamination of A, catalyzed by adenosine deaminase acting RNA (ADAR) in a process called A-to-I RNA editing. A-to-I RNA editing affects various biological processes by modulating gene expression. In addition, dysregulation of A-to-I RNA editing results in pathological consequences. I on RNA strands is converted to guanosine (G) during cDNA synthesis by reverse transcription. Thus, the conventional method used to identify A-to-I RNA editing sites compares cDNA sequences with their corresponding genomic sequences. Combined with deep sequencing, this method has been applied to transcriptome-wide screening of A-to-I RNA editing sites. This approach, however, produces a large number of false positives mainly owing to mapping errors. To address this issue, we developed a biochemical method called inosine chemical erasing (ICE) to reliably identify genuine A-to-I RNA editing sites. In addition, we applied the ICE method combined with RNA-seq, referred to as ICE-seq, to identify transcriptome-wide A-to-I RNA editing sites. In this chapter, we describe the detailed protocol for ICE-seq, which can be applied to various sources and taxa.
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Shunpei
authors.initials: S
authors.lastname: Okada
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Sakurai
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Ueda
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Bunkyo-ku, Tokyo, Japan. Electronic address: ts@chembio.t.u-tokyo.ac.jp.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Suzuki
mesh_terms: D000241:Adenosine / Q000235:genetics; D000243:Adenosine Deaminase / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D015894:Genome, Human; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007288:Inosine / Q000235:genetics; D017393:RNA Editing / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods; D059467:Transcriptome / Q000235:genetics
chemical_list: D007288:Inosine; D000243:Adenosine Deaminase; D000241:Adenosine
keywords: A-to-I RNA editing; ADAR; ICE method; ICE-seq; Inosine; RDD