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Monitoring genome-wide replication fork directionality by Okazaki fragment sequencing in mammalian cells

Nature Protocols volume 16, pages 1193–1218 (2021)Cite this article

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Abstract

The ability to monitor DNA replication fork directionality at the genome-wide scale is paramount for a greater understanding of how genetic and environmental perturbations can impact replication dynamics in human cells. Here we describe a detailed protocol for isolating and sequencing Okazaki fragments from asynchronously growing mammalian cells, termed Okazaki fragment sequencing (Ok-seq), for the purpose of quantitatively determining replication initiation and termination frequencies around specific genomic loci by meta-analyses. Briefly, cells are pulsed with 5-ethynyl-2′-deoxyuridine (EdU) to label newly synthesized DNA, and collected for DNA extraction. After size fractionation on a sucrose gradient, Okazaki fragments are concentrated and purified before click chemistry is used to tag the EdU label with a biotin conjugate that is cleavable under mild conditions. Biotinylated Okazaki fragments are then captured on streptavidin beads and ligated to Illumina adapters before library preparation for Illumina sequencing. The use of Ok-seq to interrogate genome-wide replication fork initiation and termination efficiencies can be applied to all unperturbed, asynchronously growing mammalian cells or under conditions of replication stress, and the assay can be performed in less than 2 weeks.

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Fig. 1: Schematic overview of Ok-seq protocol.
Fig. 2: Schematic representation of expected Okazaki fragment distributions for replication initiation and termination sites.
Fig. 3: Anticipated results from RPE-1 cells.
Fig. 4: Agarose gel and TapeStation results.

Data availability

Data used for the non-downsampled (100%) curves in Fig. 4 are provided at GSE114017. The downsampled curves can be produced by repeatedly removing one-half of the reads from the fastq files and then re-running the processing pipeline on the reduced dataset.

Code availability

All code is publicly available under the GNU General Public License v2.0 in our GitHub repository: https://github.com/FenyoLab/Ok-Seq_Processing. Included in the repository is example input data for creating plots as shown in Fig. 4, as well as additional plots as described in the ‘Data analysis’ section. The expected output of all scripts is also included as pdf/jpg files. Documentation is provided as a readme file, and specific instructions on parameters to functions are embedded as inline comments in the code. The current version of the software is v1.0, tagged as a release in GitHub: https://github.com/FenyoLab/Ok-Seq_Processing/releases/tag/v1.0.

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Acknowledgements

The authors thank A. Heguy and O. Zappile from the NYU Genome Technology Center for assistance with TapeStation and sequencing. Work in the T.T.H. laboratory is supported by grants from the NIH (ES025166), V Foundation BRCA Research and Basser Innovation Award. Work in D.J.S. laboratory is supported by grant (R35 GM134918) from the NIH.

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Author notes

  1. These authors contributed equally: Sarah Kit Leng Lui, Sarah Keegan.

Authors and Affiliations

  1. Department of Biochemistry & Molecular Pharmacology, New York University School of Medicine, New York, NY, USA

    Sarah Kit Leng Lui, Sarah Keegan, Peter Tonzi, Yu-Hung Chen, Noor Chalhoub, Kate E. Coleman, David Fenyo & Tony T. Huang

  2. Institute for Systems Genetics, New York University School of Medicine, New York, NY, USA

    Sarah Keegan & David Fenyo

  3. Department of Biology, New York University, New York, NY, USA

    Malik Kahli & Duncan J. Smith

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Contributions

Y.-H.C. and M.K. developed the original protocol from our labs, P.T., S.K.L.L., N.C., and K.E.C. made modifications in the DNA extraction steps and performed the experiments, and S.K., D.F., and D.J.S. provided bioinformatics support. S.K.L.L. and S.K. wrote the manuscript, S.K., D.F., D.J.S., and T.T.H. analyzed the data, and T.T.H. and D.J.S. conceived the project.

Corresponding authors

Correspondence to David Fenyo, Duncan J. Smith or Tony T. Huang.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Anja-Katrin Bielinsky, Conrad A. Nieduszynski, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Chen, Y. et al. Nat. Struct. Mol. Biol. 26, 67–77 (2019): https://doi.org/10.1038/s41594-018-0171-0

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Kit Leng Lui, S., Keegan, S., Tonzi, P. et al. Monitoring genome-wide replication fork directionality by Okazaki fragment sequencing in mammalian cells. Nat Protoc 16, 1193–1218 (2021). https://doi.org/10.1038/s41596-020-00454-5

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