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

Nature Protocols volume 12, pages 122149 (2017) | Download Citation

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.

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Acknowledgements

We thank the CellNetworks Deep Sequencing Core Facility at Heidelberg University, in particular D. Ibberson, as well as Vertis AG, in particular F. Thümmler, for Illumina sequencing and helpful discussions about adaptor and primer design. We thank J. Becker, A. Krause, A. Samanta, B. Strauß, Y.Q. Zhang, M. Tesch and other members of the Jäschke laboratory for help and discussions. M.L.W. was supported by a PhD fellowship from HBIGS. H.C. was supported by a postdoctoral fellowship from the Alexander-von-Humboldt Foundation. A.J. was supported by the German Research Council (DFG SPP 1784), the BMBF, the Helmholtz Initiative on Synthetic Biology and Baden-Württemberg Stiftung.

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Author notes

    • Marie-Luise Winz
    •  & Hana Cahová

    Present addresses: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK (M.-L.W.); Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic (H.C.).

Affiliations

  1. Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Heidelberg, Germany.

    • Marie-Luise Winz
    • , Hana Cahová
    • , Gabriele Nübel
    • , Jens Frindert
    • , Katharina Höfer
    •  & Andres Jäschke

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Contributions

All authors contributed to design of experiments; M.-L.W., H.C., G.N., J.F. and K.H. performed experiments; all authors analyzed experiments and contributed to the writing of the manuscript.

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The authors declare no competing financial interests.

Corresponding author

Correspondence to Andres Jäschke.

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https://doi.org/10.1038/nprot.2016.163

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