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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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Hauptverfasser: Winz, Marie-Luise (VerfasserIn) , Cahová, Hana (VerfasserIn) , Nübel, Gabriele (VerfasserIn) , Frindert, Jens (VerfasserIn) , Höfer, Katharina (VerfasserIn) , Jäschke, Andres (VerfasserIn)
Dokumenttyp: Article (Journal)
Sprache:Englisch
Veröffentlicht: 2017
In: Nature protocols
Year: 2016, Jahrgang: 12, Heft: 1, Pages: 122-149
ISSN:1750-2799
DOI:10.1038/nprot.2016.163
Online-Zugang: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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Verfasserangaben:Marie-Luise Winz, Hana Cahová, Gabriele Nübel, Jens Frindert, Katharina Höfer & Andres Jäschke
Beschreibung
Zusammenfassung: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.
Beschreibung:Published: 15 December 2016
Gesehen am 16.07.2018
Beschreibung:Online Resource
ISSN:1750-2799
DOI:10.1038/nprot.2016.163

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