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

Kód výsledku v 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>

Výsledek na webu

<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>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2017

Kód důvěrnosti údajů

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

Název periodika

Nature Protocols

ISSN

1754-2189

e-ISSN

—

Svazek periodika

12

Číslo periodika v rámci svazku

1

Stát vydavatele periodika

GB - Spojené království Velké Británie a Severního Irska

Počet stran výsledku

28

Strana od-do

122-149

Kód UT WoS článku

000394186700008

EID výsledku v databázi Scopus

2-s2.0-85020920270