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The establishment of silent chromatin, a heterochromatin-like structure at HML and HMR in Saccharomyces cerevisiae, depends on progression through S phase of the cell cycle, but the molecular nature of this requirement has remained elusive despite intensive study. Using highresolution chromatin immunoprecipitation and single-molecule RNA analysis, we found that silencing establishment proceeded via gradual repression of transcription in individual cells over several cell cycles, and that thedoi:10.7554/elife.58910.sa1 fatcat:fxwl6ygdbfdonc775tvidsvk7q