Department of Biochemistry & Molecular Pharmacology, New York University School of Medicine, New York, NY, USA.
Institute for Systems Genetics, New York University School of Medicine, New York, NY, USA.
Nat Protoc. 2021 Feb;16(2):1193-1218. doi: 10.1038/s41596-020-00454-5. Epub 2021 Jan 13.
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 复制叉方向的能力对于更好地理解遗传和环境干扰如何影响人类细胞的复制动力学至关重要。在这里，我们描述了一种从同步生长的哺乳动物细胞中分离和测序 Okazaki 片段的详细方案，称为 Okazaki 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）脉冲标记新合成的 DNA，并进行 DNA 提取。在蔗糖梯度上进行大小分级后，Okazaki 片段在点击化学作用下被浓缩和纯化，然后用可在温和条件下切割的生物素缀合物标记 EdU 标记。然后，生物素化的 Okazaki 片段被链霉亲和素珠捕获，并连接到 Illumina 接头，然后进行 Illumina 测序文库制备。使用 Ok-seq 来研究全基因组复制叉起始和终止效率可应用于所有未受干扰的、同步生长的哺乳动物细胞或在复制应激条件下，并且该测定可在不到 2 周的时间内完成。
