Protocol for genome-wide DNA replication timing analysis using click chemistry-based biotinylation

DNA replication timing (RT) is the cell-type-specific order by which different genomic regions are replicated during the S phase. Here, we present a biotinylation-based version of Repli-seq (BioRepli-seq) to determine genome-wide RT through next-generation sequencing. We detail steps for nucleotide analog pulse labeling, DNA content-based cell sorting, click chemistry-based biotinylation, DNA fragmentation, and on-bead sequencing library generation. This technique can be applied to any proliferating cell type to study RT and is compatible with automatization. For complete details on the use and execution of this protocol, please refer to Gökbuget et al.¹.