Mapping replication timing domains genome wide in single mammalian cells with single-cell DNA replication sequencing
- 23 November 2020
- journal article
- research article
- Published by Springer Science and Business Media LLC in Nature Protocols
- Vol. 15 (12), 4058-4100
- https://doi.org/10.1038/s41596-020-0378-5
Abstract
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.Funding Information
- MEXT | Japan Society for the Promotion of Science (JP16H01405, JP19K06610, JP18K14681, JP15H01462, JP17H06426, JP15K06942, JP18H05530)
- Special Postdoctoral Researcher (SPDR) Program of RIKEN
- RIKEN CDB/BDR intramural grant.‘Epigenome Manipulation Project’ of the All-RIKEN Projects.
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