A Versatile Procedure to Generate Genome-Wide Spatiotemporal Program of Replication in Yeast Species.
Here, we describe a complete protocol, comprising both the experimental and the analytical procedures, that allows to generate genome-wide spatiotemporal program of replication and to find the location of chromosomally active replication origins in yeast. The first step consists on synchronizing a cell population by physical discrimination of G1 cells according to their sedimentation coefficient. G1 cells are then synchronously released into S-phase and time-point samples are regularly taken until they reach the G2 phase. Progression through the cell cycle is monitored by measuring DNA content variation by flow cytometry. DNA samples, covering the entire S-phase, are then extracted and analyzed using deep sequencing. The gradual change of DNA copy number is measured to determine the mean replication time along the genome. A simple method of peak calling allows to infer from the replication profile the location of replication origins along the chromosomes. Our protocol is versatile enough to be applied to virtually any yeast species of interest and generate its replication profile.
Biologie Computationnelle et Quantitative, Sorbonne Universités, UPMC Univ Paris 06, UMR 7238, 75005, Paris, France.|Biologie Computationnelle et Quantitative, CNRS-Université Pierre et Marie Curie, UMR 7238, Site des Cordeliers, Bât. A - 4ème étage, 15, Rue de l'Ecole de Médecine, 75005, Paris, France.
Nicolas
N
Agier
Biologie Computationnelle et Quantitative, Sorbonne Universités, UPMC Univ Paris 06, UMR 7238, 75005, Paris, France. gilles.fischer@upmc.fr.|Biologie Computationnelle et Quantitative, CNRS-Université Pierre et Marie Curie, UMR 7238, Site des Cordeliers, Bât. A - 4ème étage, 15, Rue de l'Ecole de Médecine, 75005, Paris, France. gilles.fischer@upmc.fr.
Gilles
G
Fischer
D002453:Cell Cycle / Q000235:genetics; D015825:Chromosomes, Fungal / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods*; D016681:Genome, Fungal; D018741:Replication Origin / Q000235:genetics; D016196:S Phase / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics
DNA copy number; Deep sequencing; Elutriation; Flow cytometry; G1 synchronization; Gradient of Percoll; Origins; Replication; Yeast