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. Briefly, cells are pulsed with 5-ethynyl-2′-deoxyuridine (EdU) to label newly synthesized DNA, and collected for DNA extraction. 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. 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.Ībstract = "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. Biotinylated Okazaki fragments are then captured on streptavidin beads and ligated to Illumina adapters before library preparation for Illumina sequencing. Haruki H, Nishikawa J, Laemmli UK (2008) The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes.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.Haruki H, Nishikawa J, Laemmli UK (2008) The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes.Kubota T, Nishimura K, Kanemaki MT et al (2013) The Elg1 replication factor C-like complex functions in PCNA Unloading during DNA replication.Sanchez-Diaz A, Kanemaki M, Marchesi V et al (2004) Rapid depletion of budding yeast proteins by fusion to a heat-inducible degron.Rohland N, Reich D (2012) Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture.McGuffee SR, Smith DJ, Whitehouse I (2013) Quantitative, genome-wide analysis of eukaryotic replication initiation and termination.Smith DJ, Whitehouse I (2012) Intrinsic coupling of lagging-strand synthesis to chromatin assembly.Nasmyth KA (1977) Temperature-sensitive lethal mutants in the structural gene for DNA ligase in the yeast Schizosaccharomyces pombe.Johnston LH, Nasmyth KA (1978) Saccharomyces cerevisiae cell cycle mutant cdc9 is defective in DNA ligase.Distribution of functions between FEN1 AND DNA2. Ayyagari R, Gomes XV, Gordenin DA et al (2003) Okazaki fragment maturation in yeast.Waga S, Stillman B (1994) Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro.Nethanel T, Kaufmann G (1990) Two DNA polymerases may be required for synthesis of the lagging DNA strand of simian virus 40.
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