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.

在全基因组范围内监测 DNA 复制叉方向性的能力对于更好地了解遗传和环境扰动如何影响人类细胞中的复制动态至关重要。在这里，我们描述了从异步生长的哺乳动物细胞中分离和测序冈崎片段的详细协议，称为冈崎片段测序 (Ok-seq)，目的是通过元分析定量确定特定基因组位点周围的复制起始和终止频率。简而言之，细胞用 5-乙炔基-2'-脱氧尿苷 (EdU) 脉冲以标记新合成的 DNA，并收集用于 DNA 提取。在蔗糖梯度上进行尺寸分级后，在使用点击化学标记 EdU 标记之前，冈崎片段被浓缩和纯化，该标记带有在温和条件下可切割的生物素偶联物。然后在链霉亲和素珠上捕获生物素化的 Okazaki 片段并连接到 Illumina 适配器，然后为 Illumina 测序进行文库制备。使用 Ok-seq 询问全基因组复制叉的起始和终止效率可应用于所有未受干扰、异步生长的哺乳动物细胞或在复制压力条件下，并且该测定可在不到 2 周的时间内完成。