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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| Main Authors: | , , , , , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
2017
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| 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 |
MARC
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| 520 | |a 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. | ||
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