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Adenosine to inosine (A &gt; I) RNA editing, which is catalyzed by the ADAR family of proteins, is one of the fundamental mechanisms by which transcriptomic diversity is generated. Indeed, a number of genome-wide analyses have shown that A &gt; I editing is not limited to a few mRNAs, as originally thought, but occurs widely across the transcriptome, especially in the brain. Importantly, there is increasing evidence that A &gt; I editing is essential for animal development and nervous system function. To more efficiently characterize the complete catalog of ADAR events in the mammalian transcriptome we developed a high-throughput protocol to identify A &gt; I editing sites, which exploits the capacity of glyoxal to protect guanosine, but not inosine, from RNAse T1 treatment, thus facilitating extraction of RNA fragments with inosine bases at their termini for high-throughput sequencing. Using this method we identified 665 editing sites in mouse brain RNA, including most known sites and suite of novel sites that include nonsynonymous changes to protein-coding genes, hyperediting of genes known to regulate p53, and alterations to non-protein-coding RNAs. This method is applicable to any biological system for the de novo discovery of A &gt; I editing sites, and avoids the complicated informatic and practical issues associated with editing site identification using traditional RNA sequencing data. This approach has the potential to substantially increase our understanding of the extent and function of RNA editing, and thereby to shed light on the role of transcriptional plasticity in evolution, development, and cognition.

Transcriptome-wide identification of A > I RNA editing sites by inosine specific cleavage