Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

KAS-seq: genome-wide sequencing of single-stranded DNA by N3-kethoxal–assisted labeling

Abstract

Transcription and its dynamics are crucial for gene expression regulation. However, very few methods can directly read out transcriptional activity with low-input material and high temporal resolution. This protocol describes KAS-seq, a robust and sensitive approach for capturing genome-wide single-stranded DNA (ssDNA) profiles using N3-kethoxal–assisted labeling. We developed N3-kethoxal, an azido derivative of kethoxal that reacts with deoxyguanosine bases of ssDNA in live cells within 5–10 min at 37 °C, allowing the capture of dynamic changes. Downstream biotinylation of labeled DNA occurs via copper-free click chemistry. Altogether, the KAS-seq procedure involves N3-kethoxal labeling, DNA isolation, biotinylation, fragmentation, affinity pull-down, library preparation, sequencing and bioinformatics analysis. The pre-library construction labeling and enrichment can be completed in as little as 3–4 h and is applicable to both animal tissue and as few as 1,000 cultured cells. Our recent study shows that ssDNA signals measured by KAS-seq simultaneously reveal the dynamics of transcriptionally engaged RNA polymerase (Pol) II, transcribing enhancers, RNA Pol I and Pol III activities and potentially non-canonical DNA structures with high analytical sensitivity. In addition to the experimental protocol, we also introduce here KAS-pipe, a user-friendly integrative data analysis pipeline for KAS-seq.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: The chemical principles and workflow of KAS-seq.
Fig. 2: Comparison of KAS-seq with a potassium permanganate (KMnO4)-based method.
Fig. 3: Comparison between KAS-seq and other transcriptional activity–profiling methods.
Fig. 4: Quality control of KAS-seq data in bulk HEK293T cells.
Fig. 5: Anticipated results using the integrative KAS-seq data analysis pipeline (KAS-pipe).

Data availability

KAS-seq data in HEK293T and mouse embryonic stem cell lines are available at the National Center for Biotechnology Information Gene Expression Omnibus repository under the accession number GSE97072. Global run-on sequencing (GRO-seq) data in HEK293T cells are available under the accession number GSE92375. Pol II ChIP-seq and 4-thiouridine (4SU) nascent RNA-seq data in HEK293T cells are available under the accession number GSE112608.

Code availability

All the KAS-pipe code used in this study is available at https://github.com/Ruitulyu/KAS-pipe26.

References

  1. Chen, F. X., Smith, E. R. & Shilatifard, A. Born to run: control of transcription elongation by RNA polymerase II. Nat. Rev. Mol. Cell Biol. 19, 464–478 (2018).

    CAS  PubMed  Article  Google Scholar 

  2. Bell, S. P. & Dutta, A. DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374 (2002).

    CAS  PubMed  Article  Google Scholar 

  3. Hustedt, N. & Durocher, D. The control of DNA repair by the cell cycle. Nat. Cell Biol. 19, 1–9 (2017).

    CAS  Article  Google Scholar 

  4. Li, X. & Heyer, W.-D. Homologous recombination in DNA repair and DNA damage tolerance. Cell Res. 18, 99–113 (2008).

    CAS  PubMed  Article  Google Scholar 

  5. Fuchs, G. et al. 4sUDRB-seq: measuring genomewide transcriptional elongation rates and initiation frequencies within cells. Genome Biol. 15, R69 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  6. Kouzine, F. et al. Permanganate/S1 nuclease footprinting reveals non-B DNA structures with regulatory potential across a mammalian genome. Cell Syst. 4, 344–356.e7 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. Huppert, J. L. & Balasubramanian, S. G-quadruplexes in promoters throughout the human genome. Nucleic Acids Res. 35, 406–413 (2007).

    CAS  PubMed  Article  Google Scholar 

  8. Zeraati, M. et al. I-motif DNA structures are formed in the nuclei of human cells. Nat. Chem. 10, 631–637 (2018).

    CAS  PubMed  Article  Google Scholar 

  9. Cer, R. Z. et al. Non-B DB v2.0: a database of predicted non-B DNA-forming motifs and its associated tools. Nucleic Acids Res. 41, D94–D100 (2012).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  10. McIntosh, D. B., Duggan, G., Gouil, Q. & Saleh, O. A. Sequence-dependent elasticity and electrostatics of single-stranded DNA: signatures of base-stacking. Biophys. J. 106, 659–666 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Murphy, M., Rasnik, I., Cheng, W., Lohman, T. M. & Ha, T. Probing single-stranded DNA conformational flexibility using fluorescence spectroscopy. Biophys. J. 86, 2530–2537 (2004).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Ginno, P. A., Lott, P. L., Christensen, H. C., Korf, I. & Chédin, F. R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters. Mol. Cell 45, 814–825 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Sollier, J. et al. Transcription-coupled nucleotide excision repair factors promote R-loop-induced genome instability. Mol. Cell 56, 777–785 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. Lei, M., Podell, E. R. & Cech, T. R. Structure of human POT1 bound to telomeric single-stranded DNA provides a model for chromosome end-protection. Nat. Struct. Mol. Biol. 11, 1223–1229 (2004).

    CAS  PubMed  Article  Google Scholar 

  15. Zeitlin, S. G. et al. Double-strand DNA breaks recruit the centromeric histone CENP-A. Proc. Natl Acad. Sci. USA 106, 15762–15767 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. Kouzine, F. et al. Global regulation of promoter melting in naive lymphocytes. Cell 153, 988–999 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. Weng, X. et al. Keth-seq for transcriptome-wide RNA structure mapping. Nat. Chem. Biol. 16, 489–492 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. Wu, T., Lyu, R., You, Q. & He, C. Kethoxal-assisted single-stranded DNA sequencing captures global transcription dynamics and enhancer activity in situ. Nat. Methods 17, 515–523 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. Krueger, F. Trim Galore. A wrapper tool around Cutadapt and FastQC to consistently apply quality and adapter trimming to FastQ files https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/ (2015).

  20. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    PubMed  PubMed Central  Google Scholar 

  22. Feng, J., Liu, T., Qin, B., Zhang, Y. & Liu, X. S. Identifying ChIP-seq enrichment using MACS. Nat. Protoc. 7, 1728–1740 (2012).

    CAS  PubMed  Article  Google Scholar 

  23. Ramirez, F., Dundar, F., Diehl, S., Gruning, B. A. & Manke, T. deepTools: a flexible platform for exploring deep-sequencing data. Nucleic Acids Res. 42, W187–W191 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  25. Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. Lyu, R. Ruitulyu/KAS-pipe: first release of KAS-pipe for KAS-seq data analysis (1.0.0). Zenodo https://doi.org/10.5281/zenodo.4941764 (2021).

  27. Shapiro, R., Cohen, B. I., Shiuey, S.-J. & Maurer, H. Reaction of guanine with glyoxal, pyruvaldehyde, and kethoxal, and the structure of the acylguanines. Synthesis of N2-alkylguanines. Biochemistry 8, 238–245 (1969).

    CAS  PubMed  Article  Google Scholar 

  28. Staehelin, M. Inactivation of virus nucleic acid with glyoxal derivatives. Biochim. Biophys. Acta 31, 448–454 (1959).

    CAS  PubMed  Article  Google Scholar 

  29. Litt, M. & Hancock, V. Kethoxal—a potentially useful reagent for the determination of nucleotide sequences in single-stranded regions of transfer ribonucleic acid. Biochemistry 6, 1848–1854 (1967).

    CAS  PubMed  Article  Google Scholar 

  30. Noller, H. F. & Chaires, J. B. Functional modification of 16S ribosomal RNA by kethoxal. Proc. Natl Acad. Sci. USA 69, 3115–3118 (1972).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. LaGrandeur, T. E., Hüttenhofer, A., Noller, H. F. & Pace, N. R. Phylogenetic comparative chemical footprint analysis of the interaction between ribonuclease P RNA and tRNA. EMBO J. 13, 3945–3952 (1994).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. Yamane, A. et al. RPA accumulation during class switch recombination represents 5′–3′ DNA-end resection during the S–G2/M phase of the cell cycle. Cell Rep. 3, 138–147 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Lange, J. et al. The landscape of mouse meiotic double-strand break formation, processing, and repair. Cell 167, 695–708.e16 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. Paiano, J. et al. ATM and PRDM9 regulate SPO11-bound recombination intermediates during meiosis. Nat. Commun. 11, 857 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. Hinch, A. G. et al. The configuration of RPA, RAD51, and DMC1 binding in meiosis reveals the nature of critical recombination intermediates. Mol. Cell 79, 689–701.e10 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. Khil, P. P., Smagulova, F., Brick, K. M., Camerini-Otero, R. D. & Petukhova, G. V. Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA. Genome Res. 22, 957–965 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. Zhou, Z.-X. et al. Mapping genomic hotspots of DNA damage by a single-strand-DNA-compatible and strand-specific ChIP-seq method. Genome Res. 23, 705–715 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. Core, L. J., Waterfall, J. J. & Lis, J. T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845–1848 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. Paulsen, M. T. et al. Coordinated regulation of synthesis and stability of RNA during the acute TNF-induced proinflammatory response. Proc. Natl Acad. Sci. USA 110, 2240–2245 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. Fuchs, G. et al. 4sUDRB-seq: measuring genomewide transcriptional elongation rates and initiation frequencies within cells. Genome Biol. 15, R69 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  41. Barski, A. et al. High-resolution profiling of histone methylations in the human genome. Cell 129, 823–837 (2007).

    CAS  PubMed  Article  Google Scholar 

  42. Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of native chromatin for multimodal regulatory analysis and personal epigenomics. Nat. Methods 10, 1213–1218 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. Chédin, F. Nascent connections: R-loops and chromatin patterning. Trends Genet. 32, 828–838 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  44. Herschlag, D. Biophysical, Chemical, and Functional Probes of RNA Structure, Interactions and Folding: Part A (Academic Press, 2009).

  45. Akinsiku, O. T., Yu, E. T. & Fabris, D. Mass spectrometric investigation of protein alkylation by the RNA footprinting probe kethoxal. J. Mass Spectrom. 40, 1372–1381 (2005).

    CAS  PubMed  Article  Google Scholar 

  46. Kaya-Okur, H. S. et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat. Commun. 10, 1930 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  47. Laos, R., Thomson, J. M. & Benner, S. A. DNA polymerases engineered by directed evolution to incorporate non-standard nucleotides. Front. Microbiol. 5, 565 (2014).

    PubMed  PubMed Central  Article  Google Scholar 

  48. Schadt, E. E. et al. Modeling kinetic rate variation in third generation DNA sequencing data to detect putative modifications to DNA bases. Genome Res. 23, 129–141 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. Mahat, D. B. & Lis, J. T. Use of conditioned media is critical for studies of regulation in response to rapid heat shock. Cell Stress Chaperones 22, 155–162 (2017).

    PubMed  Article  Google Scholar 

  50. Cramer, P. Organization and regulation of gene transcription. Nature 573, 45–54 (2019).

    CAS  PubMed  Article  Google Scholar 

Download references

Acknowledgements

We thank all He laboratory members for discussion. We thank the Functional Genomics Facility at The University of Chicago for performing high-throughput sequencing (P30 CA014599). This work was supported by the US National Institutes of Health (R01 HG006827, RM1 HG008935 and P01 NS097206 to C.H.). C.H. is an investigator of the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

Authors

Contributions

T.W. developed experimental procedures and performed most experiments. D.C.W.-S. and X.W. validated the whole protocol. R.L. performed data analysis and developed the data analysis pipeline with suggestions from A.C.Z. M.C., R.L. and C.H. wrote the manuscript with input and edits from all authors.

Corresponding author

Correspondence to Chuan He.

Ethics declarations

Competing interests

The University of Chicago has filed a patent application on KAS-seq. C.H. is a scientific founder and a member of the scientific advisory board of Accent Therapeutics, Inc. and AccuraDX, Inc., as well as a shareholder of Epican Genetech. T.W. and D.C.W.-S. are shareholders of AccuraDX, Inc.

Additional information

Peer review information Nature Protocols thanks Fedor Kouzine, Jian Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Weng, X. et al. Nat. Chem. Biol. 16, 489–492 (2020): https://doi.org/10.1038/s41589-019-0459-3

Wu, T. et al. Nat. Methods 17, 515–523 (2020): https://doi.org/10.1038/s41592-020-0797-9

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Lyu, R., Wu, T., Zhu, A.C. et al. KAS-seq: genome-wide sequencing of single-stranded DNA by N3-kethoxal–assisted labeling. Nat Protoc 17, 402–420 (2022). https://doi.org/10.1038/s41596-021-00647-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-021-00647-6

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing