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

Sarah Kit Leng Lui, Sarah Keegan, Peter Tonzi, Malik Kahli, Yu Hung Chen, Noor Chalhoub, Kate E. Coleman, David Fenyo, Duncan J. Smith, Tony T. Huang

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)1193-1218
Number of pages26
JournalNature Protocols
Volume16
Issue number2
DOIs
StatePublished - Feb 2021

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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