(A) Phosphatase treatment of Mcm4. rif1∆ or rif1-pp1bs cells were arrested with alpha factor (G1) and then released into HU (S). Protein lysate from S phase samples was subjected to lambda phosphatase treatment, and the level of the Mcm4 shifted band was detected by western. (B) Cell cycle analysis of asynchronous, G1, and S phase arrested cells for phosphatase experiment shown in A. (C) Cell cycle analysis of asynchronous, G1, and S phase from Figure 1A was measured using flow cytometry. (D) The relative copy number for 1 kb around the midpoint of each origin was calculated for three WT strains: WT1, WT2 and WT3. For each peak, the WT value from one sequencing run was subtracted from the WT value of an independent run and multiplied by 100. Median and interquartile range are plotted over the distribution, and one-sided Wilcoxon signed rank test used to test significance: WT2-WT1 (p=0.8887), WT2-WT3 (p=1) and WT1-WT3 (p=0.9765), indicating the origin firing in the WT strains are comparable between experiments. (E) Relative copy number for the entire Chromosome VI is shown. WT is in purple, rif1∆ is in orange, and rif1-pp1bs is in blue, relating to Figure 1C. (F) The relative copy number for 1 kb around the midpoint of each origin was calculated for rif1∆ or rif1-pp1bs. For each peak, the WT value was subtracted from mutant value and multiplied by 100. A positive value indicates more origins fired in the population in the mutant compared to WT. Median and interquartile range are plotted over the distribution, and * indicates a significant difference by one-sided Wilcoxon signed rank test: rif1∆ (p<0.0001) and rif1-pp1bs (p<0.0001). (G) Heatmap of relative copy number for 10 kb region centered at each OriDB origin. Orange indicates high copy number and blue indicates low copy number. Origins sorted based on highest copy number to lowest copy number.