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

Nature Protocols volume 17, pages 402–420 (2022)Cite this article

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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.

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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).

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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.

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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.

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Author notes

  1. These authors contributed equally: Ruitu Lyu, Tong Wu.

Authors and Affiliations

  1. Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA

    Ruitu Lyu, Tong Wu, Allen C. Zhu & Chuan He

  2. Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA

    Ruitu Lyu, Tong Wu, Allen C. Zhu & Chuan He

  3. Medical Scientist Training Program, The University of Chicago, Chicago, IL, USA

    Allen C. Zhu

  4. Department of Medicine, The University of Chicago, Chicago, IL, USA

    Diana C. West-Szymanski & Mengjie Chen

  5. College of Chemistry and Molecular Sciences, Hubei Province Key Laboratory of Allergy and Immunology, Wuhan University, Wuhan, Hubei, China

    Xiaocheng Weng

  6. Department of Human Genetics, The University of Chicago, Chicago, IL, USA

    Mengjie Chen

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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.

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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.

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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

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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

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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

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