Figure 8. H3K9m2 methylation of siRNA clusters. Graph shows the percentage of different categories of siRNA clusters (dense, medium, or sparse) that overlap with H3K9m2 positive regions in either pericentromeric regions (orange) or arms (purple). doi:10.1371/journal.pone.0003156.g008 washing and scanning. For the ChIP with crosslinked tissue, a previously described protocol was used [45]. Microarray design and data analysis Together with Roche Nimblegen, we designed a forward strand Arabidopsis whole-genome array which contained ,1.98 million 60-nt oligonucleotide features. Of the ,1.98 million features, a total number of ,1.87 million features matched a unique position in the genome. Three biological replicas were performed for the MNase digested ChIP samples. Each probe was assigned a log of the ratio between H3K9m2(Cy5) and H3(Cy3) signal. Z-scores were calculated for each probe by subtracting the mean and dividing by the standard deviation for the entire array. The Z- scores for each probe were averaged among three replicas, and the correlation between replicates was high (Pearson correlation coefficient between 0.85 and 0.9). Based on the distribution of Z-scores (Figure S2), we defined a positive probe as having a score higher than 0.2, which gave a false positive rate in the chloroplast genome of 1.19%. In order to eliminate false positive signals from single probes, we defined a set of ‘‘methylated regions’’ by combining adjacent probes with scores higher than 0.2, allowing a maximal gap of 120 base pairs, and minimum run of 60 base pairs by using Integrated Genome Browser (Affymetrix). This setting allows detection of regions with a size of a mononucleosome (147 bp), but eliminates signal from single isolated probes (60 bp). TAIR7 release of Arabidopsis genome was used for annotations of genes and transposon/pseudogenes. DNA methylation data is described in [32]. Positions of tandem, inverted and interspersed repeats, as well as expression data for gene are based on [39]. siRNA cluster data is based on the study by [51]. The data is available at http://epigenomics.mcdb.ucla.edu/H3K9m2/. Two tracks ‘H3K9m2MD:ZLrToH3’ and ‘H3K9m2CL:ZLrToH3’ represent data from native and crosslinked ChIP experiments, respectively. The default display range of z-scores is 0.0 to 2.0 with a horizontal line at 1.0. Microarray data reported in the manuscript is deposited to GEO at NCBI, series accession number GSE12383 (in accordance with MIAME guidelines). Supporting Information Figure S1 MNase digestion conditions and specificity of anti- H3K9m2 antibody. (a) Nuclear DNA digested with MNase for Genome-Wide H3K9m2 Profiling various periods of time. Six-minute digestion was used for ChIP assays. (b) Western blot analysis of interaction of anti-H3K9me2 antibody with different methylated peptides. Found at: doi:10.1371/journal.pone.0003156.s001 (2.93 MB TIF) Figure S2 Distribution of H3K9m2 levels (Z-scores) for probes corresponding to each of the five Arabidopsis nuclear chromo- somes and chloroplast. Histogram shows the distribution of the numbers of probes relative to their Z-score. Black line (0.2) indicates the cut off value used for determining H3K9m2 positive probes. Found at: doi:10.1371/journal.pone.0003156.s002 (2.06 MB TIF) Figure S3 Examples of H3K9m2 patterns observed using two different techniques for chromatin immunoprecipitation, micro- coccal nuclease digested chromatin (MNase) or crosslinked and sonicated chromatin (Crosslink). Each gray bar corresponds to the z-score of an individual probe. Genes are in green and transposons are in red. Found at: doi:10.1371/journal.pone.0003156.s003 (3.10 MB TIF) Figure S4 Correlation of H3K9m2 methylation with CHG DNA methylation. Examples show the tight association between H3K9m2 positive regions (represented by grey signal), and CHG methylation as determined by whole genome bisulfite sequencing. Bright blue rectangles represent CHG methylation and green and red rectangles represent CG and CHH methylation respectively. Found at: doi:10.1371/journal.pone.0003156.s004 (2.65 MB TIF) Figure S5 Genes are devoid of H3K9m2 and CHG methylation and have high frequency of CG methylation. Examples show the lack of association between H3K9m2 positive regions (represented by grey signal), and CG only methylated regions associated with the transcribed regions of genes, as determined by whole genome bisulfite sequencing. Green rectangles represent CG methylation and blue and red rectangles represent CHG and CHH methylation respectively. Found at: doi:10.1371/journal.pone.0003156.s005 (3.24 MB TIF) Figure S6 Chromosome-wide coordinates for pericentromeric (dark) and euchromatic arm regions in megabases. Pericentro- meric regions were assigned based on the distribution of repetitive elements, genes and DNA methylation across chromosomes. Found at: doi:10.1371/journal.pone.0003156.s006 (0.33 MB TIF) Figure S7 Distribution of Z-scores of probes found in pericen- tromeric regions or euchromatic arms of the chromosomes. Data was generated by conventional (crosslinked) ChIP. Found at: doi:10.1371/journal.pone.0003156.s007 (0.39 MB TIF) Table S1 Regions previously annotated as H3K9m2 methylated and their overlap with H3K9m2 methylation found in current study. Found at: doi:10.1371/journal.pone.0003156.s008 (0.16 MB XLS) Acknowledgments We thank Xinmin Zhang, Luke Dannenberg, Leonardo Iniquez and Heather Holster (Roche NimbleGen, Inc., Madison, WI) for the array design and technical execution of array hybridization and signal processing. Author Contributions Conceived and designed the experiments: YVB XZ SEJ. Performed the experiments: YVB. Analyzed the data: YVB XZ MP SEJ. Contributed reagents/materials/analysis tools: SC MP. Wrote the paper: YVB SEJ. PLoS ONE | www.plosone.org 10 September 2008 | Volume 3 | Issue 9 | e3156