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Discovering A-to-I RNA Editing Through Chemical Methodology "ICE-seq".

Methods in molecular biology (Clifton, N.J.) (2020-07-31)
Masayuki Sakurai, Shunpei Okada, Hiroki Ueda, Yuxi Yang
ABSTRACT

RNA editing of adenosines to inosines contributes to a wide range of biological processes by regulating gene expression post-transcriptionally. To understand the effect, accurate mapping of inosines is necessary. The most conventional method to identify an editing site is to compare the cDNA sequence with its corresponding genomic sequence. However, this method has a high false discovery rate because guanosine signals, due to experimental errors or noise in the obtained sequences, contaminate genuine inosine signals detected as guanosine. To ensure high accuracy, we developed the Inosine Chemical Erasing (ICE) method to accurately and biochemically identify inosines in RNA strands utilizing inosine cyanoethylation and reverse transcription-PCR. Furthermore, we applied this technique to next-generation sequencing technology, called ICE-seq, to conduct an unbiased genome-wide screening of A-to-I editing sites in the transcriptome.

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Millipore
Ultrafree® Centrifugal Filter, 0.5 mL Sample Volume, pore size 0.45 μm, PVDF membrane (hydrophilic)