 at the mbs1 and ade6–3049 DSB Hotspots but Not in the ura1 Cold Region
(A) Haploid strain GP6013 (rec12–201::6His-2FLAG ade6–3049) was induced for meiosis, and sonicated chromatin was prepared at the indicated time. Anti-FLAG precipitates (Rec12-ChIP) and unfractionated DNA (WCE) were analyzed by PCR with primers specific for the indicated loci (Table S4). The PCR products were analyzed by agarose gel electrophoresis and staining with ethidium bromide. Experiments were done three to five times with comparable results. In these experiments DNA replication, measured by flow-cytometry, occurred between 2 and 3 h after induction (unpublished data).
(B) Diploid strains GP6203 (rec12–201::6His-2FLAG) and GP6204 (rec12–202::6His-2HA) were induced for meiosis and analyzed as above. The relative amounts of Rec12-ChIP used for the PCR analysis with mbs1-specific primers are indicated.
(C) Diploid strains GP6203 and GP6233 (rec10–175::kanMX6 rec12–201::6His-2FLAG ade6–3049) were analyzed with the indicated primers as above. The relative amounts of WCE and Rec12-ChIP are indicated.
(D) WCE from the experiments in (C) were assayed with primers specific for the ade6 and ura1 loci, using the relative amounts of WCE indicated. Each panel includes size markers decreasing from 500 bp (black caret) in steps of 100 bp downward.
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Figure S2. Comparison of Microarray Data Corresponding to the NotI Fragment J on Chromosome I Analyzed in Different Ways
Data are the IP:WCE hybridization ratios obtained with haploid strain GP6013, before induction (blue line) and 5 h after induction (red line; mean of two experiments). Chromosome positions represent distance from the left (sequenced) end of Chromosome I.
(A) Raw data.
(B) Seven-probe sliding window. For each point the mean of the seven values centered on that point is plotted.
(C) Three-probe filter. Values >2 were replaced with values of 1 unless occurring in groups of at least three contiguous probes.
(D) Normalization using uninduced (0 h) values. For each point the 5-h value was divided by the 0-h value.
(2.8 MB PDF)
Figure S3. Quantile-Quantile Plots of IP:WCE Hybridization Ratios (log2) for Chromosome I from the Haploid Experiments (Strain GP6013) against Simulated Normal Values (with a Mean of 0 and Variance of 1)
Uninduced (0 h) data are from haploid experiment 2; induced (5 h) data are the mean of two haploid experiments. Normally distributed IP:WCE hybridization ratios (log2) should result in a straight line passing through the origin.
(A) 0 h, raw data.
(B) 0 h, filtered to remove isolated (single and double) linear values >2 (i.e., log2 > 1), identified as in Figure S2C. Note that these filtered data are nearly normally distributed.
(C) 5 h, raw data.
(D) 5 h, data filtered using criteria from Figure S2C. Note that the 5-h data have many values clearly above the normal distribution that persist after filtering, as expected for genuine Rec12-enrichment of DNA at linkage sites. The percent of total data with an abscissa value ≥1 is indicated for each plot.
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Figure S4. Quantile-Quantile Plots of IP:WCE Hybridization Ratios (log2) for Chromosome I from the Diploid Experiment (Strain GP6203) against Simulated Normal Values (with a Mean of 0 and Variance of 1)
Normally distributed IP:WCE hybridization ratios (log2) should result in a straight line passing through the origin.
(A) 0 h, raw data.
(B) 0 h, filtered to remove isolated (single and double) linear values >2 (i.e., log2 > 1), identified as in Figure S2C. Note that these filtered data have some values above the normal distribution.
(C) 5 h, raw data.
(D) 5 h, data filtered using criteria from Figure S2C. Note that the 5-h data have many values clearly above the normal distribution that persist after filtering, as expected for genuine Rec12-enrichment of DNA at linkage sites. The percent of total data with an abscissa value ≥1 is indicated for each plot.
(1.8 MB PDF)
Figure S5. Distribution of Rec12-DNA Linkages in Two Haploid Inductions (Strain GP6013) and One Diploid Induction (Strain GP6203) Are Similar
Data