bed  and then harvested, washed, and stored at −20 °C. Chromatin was prepared, sonicated to yield DNA 0.5–1 kb long as determined by agarose gel electrophoresis (unpublished data), and subjected to immunoprecipitation with anti-FLAG antibody (Sigma, http://www.sigmaaldrich.com) as described . Precipitates (IP) were washed, treated with proteinase K, extracted with phenol-CHCl3, and treated with RNase; the residual DNA was purified on a QIAquick column (Qiagen, http://www1.qiagen.com), amplified in the presence of amino-allyl dUTP, and labeled with Cy5 as described . DNA from unfractionated, sonicated chromatin (WCE) was prepared and labeled with Cy3 in parallel. The samples were mixed and hybridized to an Agilent 44-K S. pombe oligonucleotide microarray and analyzed as described . The IP:WCE ratio for each probe on the array (∼41,000) was then calculated after adjustment of the IP values to equalize the IP and WCE medians. Data were analyzed with Excel (Microsoft, http://www.microsoft.com), PeakFinder , and ChIPOTle  software. Peakfinder was run using the haploid and diploid datasets after noise reduction as in Figure 1, with linear plotting, Gaussian weighting, a single round using a 13-probe window, right and left minimum smoothed delta = 100, and a threshold of 2,000. ChIPOTle was run on the haploid dataset with noise reduction as in Figure S3. The Gaussian background setting was used with a window size of 1 kb, a step size of 150 bp, and a cutoff of p < 0.001.
Dataset S1. Median-Normalized Genome-Wide IP/WCE Hybridization Ratios from Two Meiotic Inductions of Haploid Strain GP6013
Shown are experiment 2 data from before induction (0 h) and 5 h after induction (5 h) and experiment 1 data from 5 h after induction (5 h).
(6.7 MB XLS)
Dataset S2. Median-Normalized Genome-Wide IP/WCE Hybridization Ratios from a Meiotic Induction of Diploid Strain GP6203
Shown are data from before induction (0 h) and 5 h after induction (5 h).
(6.0 MB XLS)
Figure S1. Meiosis-Specific Rec12-DNA Linkage Requires Rec10 and Is Abundant 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.
(648 KB PDF)
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 val