| Online-Ressource |
Verfasst von: | Winz, Marie-Luise [VerfasserIn]  |
| Cahová, Hana [VerfasserIn]  |
| Nübel, Gabriele [VerfasserIn]  |
| Frindert, Jens [VerfasserIn]  |
| Höfer, Katharina [VerfasserIn]  |
| 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 |
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. |
DOI: | doi:10.1038/nprot.2016.163 |
URL: | Bitte beachten Sie: Dies ist ein Bibliographieeintrag. Ein Volltextzugriff für Mitglieder der Universität besteht hier nur, falls für die entsprechende Zeitschrift/den entsprechenden Sammelband ein Abonnement besteht oder es sich um einen OpenAccess-Titel handelt.
Volltext ; Verlag: http://dx.doi.org/10.1038/nprot.2016.163 |
| Volltext: https://www.nature.com/articles/nprot.2016.163 |
| DOI: https://doi.org/10.1038/nprot.2016.163 |
Datenträger: | Online-Ressource |
Sprache: | eng |
K10plus-PPN: | 1577642430 |
Verknüpfungen: | → Zeitschrift |
Capture and sequencing of NAD-capped RNA sequences with NAD captureSeq / Winz, Marie-Luise [VerfasserIn]; 2017 (Online-Ressource)