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Single-cell DNA replication sequencing (scRepli-seq): a method for mapping replication timing domains genome wide in single mammalian cells

ABSTRACT: Single-cell DNA replication sequencing (scRepli-seq): a method for mapping replication timing domains genome wide in single mammalian cells

PROVIDER: PRJNA576643 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10251052_1.fastq.gz	Fastqsanger.gz
	SRR10251052_2.fastq.gz	Fastqsanger.gz
	SRR10251053_1.fastq.gz	Fastqsanger.gz
	SRR10251053_2.fastq.gz	Fastqsanger.gz
	SRR10251054.fastq.gz	Fastqsanger.gz

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Single-cell DNA replication sequencing (scRepli-seq): a method for mapping replication timing domains genome wide in single mammalian cells

Project description: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.

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scRepli-seq analysis of SCNT 4-cell and 8-cell embryos

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Genome-wide stability of DNA replication program in single mammalian cells

Project description:We report the establishment of a single-cell DNA replication sequencing method, scRepli-seq, which is a simple genome-wide methodology that measures copy number differences between replicated and unreplicated DNA. Using scRepli-seq, we demonstrate that replication domain organization is conserved among individual mouse embryonic stem cells (mESCs). Differentiated mESCs exhibited distinct replication profiles, which were conserved from cell to cell. Haplotype-resolved scRepli-seq revealed similar replication timing profiles of homologous autosomes, while the inactive X chromosome was clearly replicated later than its active counterpart. However, a small degree of cell-to-cell replication timing heterogeneity was present, and we discovered that developmentally regulated domains are a source of such variability, suggesting a link between cell-to-cell heterogeneity and developmental plasticity. Together, our results form a foundation for single-cell-level understanding of DNA replication regulation and provide insights into 3D genome organization.

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Rif1 regulates the replication timing domains on the human genome

Project description:DNA replication is spatially and temporally regulated during S-phase. DNA replication timing is established in early G1-phase at a point referred to as TDP (timing decision point). We show that Rif1 (Rap1-interacting-factor1), originally identified as a telomere binding factor in yeast, is a critical determinant of the replication timing program in human cells. Depletion of Rif1 results in specific loss of mid-S replication foci profiles, stimulation of initiation events in early S-phase and changes in long-range replication timing domain structures. Overall replication timing is shifted toward mid-S in both directions, suggesting that replication timing regulation is abrogated in the absence of Rif1. Rif1 tightly binds to nuclear insoluble structures at late-M to early-G1 and regulates the chromatin-loop sizes. Furthermore, Rif1 colocalizes specifically with the mid-S replication foci. Thus, Rif1 establishes the mid-S replication domains that are restrained from being activated at early S-phase. Our results indicate that Rif1 plays crucial roles in determining the replication timing domain structures through regulating higher-order chromatin architecture. HeLa cells (ATCC), with a total of 4 individual replicates

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Rif1 regulates the replication timing domains on the human genome

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Project description:This study demonstrates the stochastic nature of DNA replication-timing regulation by measures genome-wide replication timing in single-cells

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Single-cell replication profiling reveals stochastic regulation of the mammalian replication-timing program [SEQXE]

Project description:This study demonstrates the stochastic nature of DNA replication-timing regulation by measuring genome-wide replication timing in single-cells

2017-12-19 | GSE102074 | GEO

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Project description:This study demonstrates the stochastic nature of DNA replication-timing regulation by measures genome-wide replication timing in single-cells

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