Replication timing (RT) domains are stable units of chromosome structure that are regulated in the context of development and disease. Conventional genome-wide RT mapping methods require a large number of S-phase cells for either effective enrichment of replicating DNA through BrdU immunoprecipitation or the determination of copy number differences during S-phase, which restricts their application to non-abundant cell types. Here, we provide a simple, cost-effective, and robust protocol for single-cell DNA replication sequencing (scRepli-seq). The scRepli-seq methodology relies on the whole-genome amplification (WGA) of genomic DNA (gDNA) from single S-phase cells and next-generation sequencing (NGS)-based determination of copy number differences that arise between replicated and unreplicated DNA. We also provide computational pipelines that are key components of the methodology. NGS library preparation takes 3 d.
Overall design
Single-cell Repli-seq experiments in hTERT-RPE1. We deposited single-cell data sets passed (or not passed) by our MAD score-based quality control. Total 26 samples.
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