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

• Verfasst von: Winz, Marie-Luise [VerfasserIn]
• Verfasst von: Cahová, Hana [VerfasserIn]
• Verfasst von: Nübel, Gabriele [VerfasserIn]
• Verfasst von: Frindert, Jens [VerfasserIn]
• Verfasst von: Höfer, Katharina [VerfasserIn]
• Verfasst von: Jäschke, Andres [VerfasserIn]
• Titel: Capture and sequencing of NAD-capped RNA sequences with NAD captureSeq
• Verf.angabe: Marie-Luise Winz, Hana Cahová, Gabriele Nübel, Jens Frindert, Katharina Höfer & Andres Jäschke
• Jahr: 2017
• Jahr des Originals: 2016
• Umfang: 28 S.
• Fussnoten: Published: 15 December 2016 ; Gesehen am 16.07.2018
• Titel Quelle: Enthalten in: Nature protocols
• Ort Quelle: Basingstoke : Nature Publishing Group, 2006
• Jahr Quelle: 2017
• Band/Heft Quelle: 12(2017), 1, Seite 122-149
• ISSN Quelle: 1750-2799
• DOI: doi:10.1038/nprot.2016.163
• Datenträger: Online-Ressource
• Sprache: eng
• K10plus-PPN: 1577642430

Abstract

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.