title: Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement.
abstract: Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge. This article describes in detail the "Copy Number Ratio of S/G1 for mapping genomic Time of Replication" method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.
authors.affiliation: Dept. of Microbiology and Molecular Genetics, IMRIC, Faculty of Medicine, Hebrew University of Jerusalem.
authors.forename: Yishai
authors.initials: Y
authors.lastname: Yehuda
authors.affiliation: Dept. of Microbiology and Molecular Genetics, IMRIC, Faculty of Medicine, Hebrew University of Jerusalem.
authors.forename: Britny
authors.initials: B
authors.lastname: Blumenfeld
authors.affiliation: The Core Research Facility, IMRIC, Faculty of Medicine, Hebrew University of Jerusalem.
authors.forename: Dan
authors.initials: D
authors.lastname: Lehmann
authors.affiliation: Dept. of Microbiology and Molecular Genetics, IMRIC, Faculty of Medicine, Hebrew University of Jerusalem; itamarsi@ekmd.huji.ac.il.
authors.forename: Itamar
authors.initials: I
authors.lastname: Simon
mesh_terms: D000818:Animals; D019295:Computational Biology; D004247:DNA / Q000235:genetics; D042522:DNA Replication Timing; D005434:Flow Cytometry; D016193:G1 Phase; D023281:Genomics; D006801:Humans; D051379:Mice; D011003:Ploidies; D016196:S Phase
chemical_list: D004247:DNA