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Genome-wide mapping of embedded ribonucleotides and other noncanonical nucleotides using emRiboSeq and EndoSeq

Nature Protocols volume 10, pages 1433–1444 (2015)Cite this article

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Abstract

Ribonucleotides are the most common noncanonical nucleotides incorporated into the genome of replicating cells. They are efficiently removed by ribonucleotide excision repair initiated by RNase H2 cleavage. In the absence of RNase H2, such embedded ribonucleotides can be used to track DNA polymerase activity in vivo. To determine their precise location in Saccharomyces cerevisiae, we developed embedded ribonucleotide sequencing (emRiboSeq), which uses recombinant RNase H2 to selectively create ligatable 3′-hydroxyl groups, in contrast to alternative methods that use alkaline hydrolysis. EmRiboSeq allows reproducible, strand-specific and potentially quantitative detection of embedded ribonucleotides at single-nucleotide resolution. For the genome-wide mapping of other noncanonical bases, RNase H2 can be replaced with specific nicking endonucleases in this protocol; we term this method endonuclease sequencing (EndoSeq). With the protocol taking <5 d to complete, these methods allow the in vivo study of DNA replication and repair, including the identification of replication origins and termination regions.

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Figure 1: The emRiboSeq protocol.
Figure 2: Library quality control and anticipated results.

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Acknowledgements

We thank A. Gallacher for technical assistance throughout the development of this protocol and T. Kunkel (National Institute of Environmental Health Sciences) for sharing yeast strains. This work was supported by funding from the Medical Research Council (MRC; Centenary Award to M.A.M.R.), the MRC and Medical Research Foundation (to M.S.T.) and the MRC and Lister Institute of Preventive Medicine (to A.P.J.).

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  1. James Ding

    Present address: Present address: Arthritis Research UK Centre for Genetics and Genomics, Institute for Inflammation and Repair, Manchester Academic Health Science Centre, The University of Manchester, Manchester, UK.,

Authors and Affiliations

  1. Medical Research Council (MRC) Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh, UK

    James Ding, Martin S Taylor, Andrew P Jackson & Martin A M Reijns

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Contributions

M.A.M.R., M.S.T. and A.P.J. conceived and designed the original protocol. M.A.M.R. and J.D. modified and updated the protocol to its current state. M.S.T. performed all computational analyses. J.D. and M.A.M.R. wrote the manuscript with assistance from M.S.T. and A.P.J.

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Correspondence to Martin A M Reijns.

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Ding, J., Taylor, M., Jackson, A. et al. Genome-wide mapping of embedded ribonucleotides and other noncanonical nucleotides using emRiboSeq and EndoSeq. Nat Protoc 10, 1433–1444 (2015). https://doi.org/10.1038/nprot.2015.099

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