Mapping replication timing domains genome wide in single mammalian cells with single-cell DNA replication sequencing
Mapping replication timing domains genome wide in single mammalian cells with single-cell DNA replication sequencing
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DOI：
10.1038/s41596-020-0378-5
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发表时间：
2020-11-23
期刊：
影响因子：
14.8
通讯作者：
Takebayashi, Shin-ichiro
中科院分区：
文献类型：
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作者：
Miura, Hisashi;Takahashi, Saori;Takebayashi, Shin-ichiro
该方案描述了获得全基因组DNA复制时序图的实验和计算程序的基础上的拷贝数差异来自全基因组扩增和下一代测序的基因组DNA从单个S-phase cells.Replication timing（RT）域是稳定的染色体结构单位，在发展和疾病的背景下进行调节。传统的全基因组RT定位方法需要许多S期细胞，用于通过溴脱氧尿苷（BrdU）免疫沉淀有效富集复制DNA或测定S期期间的拷贝数差异，这排除了它们对非丰富细胞类型和单细胞的应用。在这里，我们为单细胞DNA复制测序（scd-seq）提供了一个简单，经济高效和强大的协议。scd-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测定。单倍型分辨scd-seq通过使用单核苷酸多态性（SNP）/indel信息来区分单个细胞内的同源染色体对是可行的。我们还提供了用于scd-seq数据的质量控制、标准化和二值化的计算管道。该方案的实验部分（测序前）需要3d。
This protocol describes experimental and computational procedures for obtaining genome-wide DNA replication timing maps based on copy-number differences derived from whole-genome amplification and next-generation sequencing of genomic DNA from single S-phase cells.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.