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 many S-phase cells for either the effective enrichment of replicating DNA through bromodeoxyuridine (BrdU) immunoprecipitation or the determination of copy-number differences during S-phase, which precludes their application to non-abundant cell types and single cells. Here, we provide a simple, cost-effective, and robust protocol for single-cell DNA replication sequencing (scRepli-seq). The scRepli-seq methodology relies on 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. Haplotype-resolved scRepli-seq, which distinguishes pairs of homologous chromosomes within a single cell, is feasible by using single-nucleotide polymorphism (SNP)/indel information. We also provide computational pipelines for quality control, normalization, and binarization of the scRepli-seq data. The experimental portion of this protocol (before sequencing) takes 3 d.

复制时间 (RT) 结构域是染色体结构的稳定单位，在发育和疾病的背景下受到调节。传统的全基因组 RT 作图方法需要许多 S 期细胞，以通过溴脱氧尿苷 (BrdU) 免疫沉淀有效富集复制 DNA 或确定 S 期期间的拷贝数差异，这排除了它们对非丰富细胞类型的应用和单细胞。在这里，我们为单细胞 DNA 复制测序 (scRepli-seq) 提供了一个简单、经济高效且强大的协议。scRepli-seq 方法依赖于来自单个 S 期细胞的基因组 DNA (gDNA) 的全基因组扩增 (WGA) 和基于下一代测序 (NGS) 的复制和未复制 DNA 之间出现的拷贝数差异的确定。通过使用单核苷酸多态性 (SNP)/indel 信息，单倍型解析的 scRepli-seq 可以区分单个细胞内的同源染色体对。我们还为 scRepli-seq 数据的质量控制、标准化和二值化提供计算管道。该协议的实验部分（测序前）需要 3 天。