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

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388963%3A_____%2F17%3A00474962" target="_blank" >RIV/61388963:_____/17:00474962 - isvavai.cz</a>

Result on the web

<a href="http://dx.doi.org/10.1038/nprot.2016.163" target="_blank" >http://dx.doi.org/10.1038/nprot.2016.163</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1038/nprot.2016.163" target="_blank" >10.1038/nprot.2016.163</a>

Result language

angličtina

Original language name

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

Original language description

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 (RTRT) 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 similar to 7 d, excluding preparatory steps, sequencing and bioinformatic analysis.

Czech name

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Czech description

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Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

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OECD FORD branch

10608 - Biochemistry and molecular biology

Project

—

Continuities

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Publication year

2017

Confidentiality

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Name of the periodical

Nature Protocols

ISSN

1754-2189

e-ISSN

—

Volume of the periodical

12

Issue of the periodical within the volume

1

Country of publishing house

GB - UNITED KINGDOM

Number of pages

28

Pages from-to

122-149

UT code for WoS article

000394186700008

EID of the result in the Scopus database

2-s2.0-85020920270