Tiling analysis software

After germination of heterozygous rscΔ strains, the ploidy of each strain was monitored by flow cytometry (Fig. 1 b). Genomic DNA from the sample that contained >95% diploid cells with the lowest number of generations was isolated. Genomic DNA from wild-type cells arrested in G₁ with α-factor was used as the control sample. CGH was performed essentially as described previously (Dion and Brown, 2009 (link); Chambers et al., 2012 (link)). In brief, genomic DNA was amplified by using a WGA2 kit (Sigma) and treated with DNaseI (NEB) to fragment DNA to a mean 50-bp length. Then, DNA fragments were labeled with biotin-N⁶-ddATP (Enzo Life Sciences) by using terminal deoxynucleotidyl transferase (Fermentas), hybridized to an S. cerevisiae Tiling Array (Affymetrix), and visualized using streptavidin R-phycoerythrin conjugate (Invitrogen) and normal goat IgG (Sigma). Experiment signal intensities from each microarray were compared with the control sample using Tiling Analysis Software (Affymetrix) using quantile normalization, perfect match probes only, a bandwidth of 60, maximum gap of 80, and minimum run of 40. The CGH profiles were generated with IGB 6.3 (Affymetrix).

Affymetrix Tiling Analysis software (Affymetrix, Inc.) and Integrated Genome Browser were used to select the positive intervals that satisfied the two conditions: that the signal intensity of the ZEB1-binding DNA and the intensity ratio of the ZEB1-binding DNA signal/normal rabbit IgG-binding DNA signal were within the top 1% among all the intervals. The filter criteria ruled out the false-positive regions with a high intensity ratio that resulted from the division of two infinitely small numbers. The positive intervals of each chromosome in bed files were uploaded to the GALAXYP online platform (usegalaxyp.org) to produce a tail-to-head concatenation and then submitted to the Cis-regulatory Element Annotation System (CEAS) to produce nearby gene mapping and motif identification. For each positive ChIP region, CEAS identified the nearest RefSeq gene names and predicted locations within 300 kb upstream or downstream of the gene. Regions within 1 kb upstream from the gene 5′ start site were assumed to be proximal promoters; those within 1 kb downstream from the gene 3′-end were reported to be immediate downstream regulatory elements; while those distributed >1 kb from the gene were assumed to be enhancers (20 (link)).

Raw microarray data files (.cel format) were normalized using either R or Affymetrix Tiling Analysis Software (TAS). TAS was used to generate log2 ratios of Fft2‐myc or Fft3‐myc to a control no‐epitope myc‐ChIP (Hu303) using two‐sample analysis quantile normalization together and a bandwidth of 100. Probe signals were assigned to S. pombe genome coordinates (Sanger 2007). TAS was also used to generate log2 ratios of mutant to WT expression arrays using the same normalization. Data analysis was performed in R (http://www.r-project.org) using the Bioconductor (http://www.bioconductor.org) packages “affy”, “affxparser”, and “preprocessCore” with standard parameters. CEL‐files were imported and quantile normalized as described in 73.
Data was visualized using Podbat 33, R, and the Integrated Genome Browser (IGB, Affymetrix). R was used to generate average gene occupancy graphs. Box‐and‐whisker plots were created in R using the “boxplot” function with standard parameters. Significance tests between data subsets were performed using the Wilcoxon–Mann–Whitney test function “wilcox.test” with standard parameters.