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

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

SUBMITTER: Kit Leng Lui S 

PROVIDER: S-EPMC8792808 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature