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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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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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.
The authors declare no competing interests.
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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Key reference using this protocol
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