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Capture and sequencing of NAD-capped RNA sequences with NAD captureSeq

Here we describe a protocol for NAD captureSeq that allows for the identification of nicotinamide-adenine dinucleotide (NAD)-capped RNA sequences in total RNA samples from different organisms. NAD-capped RNA is first chemo-enzymatically biotinylated with high efficiency, permitting selective capture...

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Bibliographic Details
Main Authors: Winz, Marie-Luise (Author) , Cahová, Hana (Author) , Nübel, Gabriele (Author) , Frindert, Jens (Author) , Höfer, Katharina (Author) , Jäschke, Andres (Author)
Format: Article (Journal)
Language:English
Published: 2017
In: Nature protocols
Year: 2016, Volume: 12, Issue: 1, Pages: 122-149
ISSN:1750-2799
DOI:10.1038/nprot.2016.163
Online Access:Verlag, Volltext: http://dx.doi.org/10.1038/nprot.2016.163
Verlag, Volltext: https://www.nature.com/articles/nprot.2016.163
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Author Notes:Marie-Luise Winz, Hana Cahová, Gabriele Nübel, Jens Frindert, Katharina Höfer & Andres Jäschke
Description
Summary:Here we describe a protocol for NAD captureSeq that allows for the identification of nicotinamide-adenine dinucleotide (NAD)-capped RNA sequences in total RNA samples from different organisms. NAD-capped RNA is first chemo-enzymatically biotinylated with high efficiency, permitting selective capture on streptavidin beads. Then, a highly efficient library preparation protocol tailored to immobilized, 5′-modified RNA is applied, with adaptor ligation to the RNA's 3′ terminus and reverse transcription (RT) performed on-bead. Then, cDNA is released into solution, tailed, ligated to a second adaptor and PCR-amplified. After next-generation sequencing (NGS) of the DNA library, enriched sequences are identified by comparison with a control sample in which the first step of chemo-enzymatic biotinylation is omitted. Because the downstream protocol does not necessarily rely on NAD-modified but on 'clickable' or biotin-modified RNA, it can be applied to other RNA modifications or RNA-biomolecule interactions. The central part of this protocol can be completed in ∼7 d, excluding preparatory steps, sequencing and bioinformatic analysis.
Item Description:Published: 15 December 2016
Gesehen am 16.07.2018
Physical Description:Online Resource
ISSN:1750-2799
DOI:10.1038/nprot.2016.163

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