SNEP identification To screen for SNEPs, we considered only pairs of aligned nucleosomes sharing at least 15 microarray probes, which was the case of 97% of aligned pairs. Following previous linear models validated for transcripts quantification [56], we applied to each pair the following analysis of variance (ANOVA): yijkl~ u z ai z bj z ck z dij z eijkl where yijkl is the log2 normalized hybridization intensity of probe k in replicate l for strain i (BY or RM) in experiment type j (nucleosome positioning or ChIP-ChIP), u is the global mean of the signal, ai is the strain effect (BY or RM), bj is the experiment type effect (nucleosome positioning or ChIP-Chip), ck is the probe effect, dij is the interaction term between strain and experiment type and eijkl is the residual. We reasoned that if a nucleosome carries the modification then the corresponding DNA is present in both ChIP and nucleosomal positioning samples and signal expectancies should not differ between experiment types (bj = 0). However, if a nucleosome carries the modification in only one strain, then a significant interaction should be seen between experiment type and strain (dij ? 0) (Figure S12). We therefore used an F-statistic to test (H0: dij = 0 vs. HA:dij ?0) and derived nucleosome-level P-values. A striking enrichment of low P-values was observed (Figure S13). We applied the false discovery rate (FDR) control procedure [28] to compute a genome-wide cutoff from our sorted vector of 58,694 P-values. Data accession numbers EMBL ArrayExpress accession number E-MEXP-1777. Pro- cessed data files and the C source code of NucleoMiner (for Unix- based platforms) are available on our web site http://www.ens-lyon. fr/LBMC/gisv/snep/ Supporting Information Figure S1 Nucleosome alignment conservation. For 63,706 pairs of BY/RM aligned nucleosomes, we computed the distance between the two midpoints (black histogram and axis), as well as the fraction of the BY nucleosome that overlapped the RM nucleosome (red histogram and axis). Found at: doi:10.1371/journal.pgen.1000913.s001 (0.22 MB AI) Figure S2 The fraction of nucleosomal DNA was computed in 2.5-Kb sliding windows across the genome, excluding windows containing insertions or deletions of 5 bp or more. (A) Histogram of the distribution of BY - RM occupancy differences among all windows. Most differences are small, and few extremes are detected. (B) Genomic location of windows with extreme values (first and last 1/1000 quantile, red = greater than 0.82, blue = lower than -0.85). Found at: doi:10.1371/journal.pgen.1000913.s002 (0.35 MB AI) Figure S3 Global nucleosomal occupancy at TSS and TES. Neighborhood regions of TSS (left panels) and TES (right panels) were divided in 10 bp bins. Probes were assigned to bins according to the position of their midpoint. For all probes, the HMM posterior probabilities to be in a nucleosomal state (delocalized or well- positioned) were summed. For every bin, this cumulated probability was averaged across all probes of the bin. Profiles shown are average profiles across all genes for BY (upper panels) and RM (lower panels). Note that coordinates of transcripts were determined in the BY strain background [23] and may slightly differ in RM. This is likely to explain the fact that depletions at TSS and TES, as well as oscillations downstream TSS appear less pronounced in RM than in BY. Found at: doi:10.1371/journal.pgen.1000913.s003 (0.29 MB AI) Single Nucleosome Epi-Polymorphisms Figure S4 The degree of acetylation differences in SNEPs is shown by their cumulative distribution of ‘foldchange’ values, which correspond to (acBY/nucBY)/(acRM/nucRM) for BYac SNEPs and its inverse for RMac SNEPs. Most SNEPs show folchanges between 1.2 and 1.5, and BYac SNEPs are associated with higher acetylation differences than RMac SNEPs. Found at: doi:10.1371/journal.pgen.1000913.s004 (0.20 MB AI) Figure S5 Scheme of nucleosome organization in the region of the NDE2 gene. Rectangles represent nucleosomes, colored according to the mean log(ac/nuc) value across all probes of the nucleosome. SNEP detection (-log10(P-value)) is indicated above each nucleosome, with significance cutoff indicated as a dashed line. Brown boxes, coding sequences. Found at: doi:10.1371/journal.pgen.1000913.s005 (0.27 MB AI) Figure S6 For all panels, BYac and RMac SNEP frequencies were counted among nucleosomes located in each gene (coding region plus and minus 250 bp), and averaged in 500-genes sliding windows (y-axis). Genes were sorted (x-axis) either by their BY/ RM foldchange in expression, which showed SNEP enrichments at both extremities (A), or by their Expression Divergence (Tirosh et al. [36]) (B), or by their mutational variance (Vm values of Landry et al. [37]) (C), which showed SNEP enrichment at high values. Found at: doi:10.1371/journal.pgen.1000913.s006 (0.42 MB AI) Figure S7 For every gene, we considered the P-value (signifi- cance) of the genotype x interaction term of the ANOVA model of Smith & Kruglyak 2008 [38]. We then ranked all genes according to sg * P, where sg is the sign of the interaction term. These ranks are reported on the x-axis. SNEP frequency (y-axis) was computed in sliding windows as in Figure S6. Found at: doi:10.1371/journal.pgen.1000913.s007 (0.29 MB AI) Figure S8 Kinetics of mRNA expression during heat-shock for three genes lacking SNEPs. Three genes (SSA3, FES1, and CPR6) were selected based on the following criteria: known to be induced upon heat-shock (from the data of Gasch et al. 2000 Mol. Biol. Cell. 11: 4241–4257), possess an HSF1 binding site in the promoter with no BY/RM polymorphism in it, do not have H3K14ac SNEP, do not have a marked BY/RM difference in expression in non-induced cultures. RNA levels of these three genes were measured by real-time quantitative RT-PCR on the same samples as for AHA1 on Figure 4. Bars: 6 standard deviations. Found at: doi:10.1371/journal.pgen.1000913.s008 (0.25 MB AI) Figure S9 Properties of SNEPs disagreeing with the patterns of Figure 2D. To distinguish SNEPs that support the general patterns of Figure 2D (BYac SNEP around TSS and TES and RMac SNEPs within transcribed region), three regions were defined. R1: from 2500 bp to TSS, R2: from TSS to TES and R3: from TES to +400 bp. We then flagged all BYac SNEP falling in R1 and R3, and all RMac SNEPs falling in R2. For nucleosomes that did not fall entirely in the region, we flagged them if .75% of their length overlapped the region. 1,806 SNEPs remained unflagged and were used to re-draw the main figures of the article. Please see the main figures of the article (that reflect all SNEPs) for legends. Found at: doi:10.1371/journal.pgen.1000913.s009 (1.91 MB AI) Figure S10 Absence of correlation between H3K14 acetylation differences and amplitudes of gene expression differences. The display is similar as in Figure 3A, except that expression fold changes are presented in the x-axis instead of statistical significance of expression differences. Found at: doi:10.1371/journal.pgen.1000913.s010 (1.47 MB AI) PLoS Genetics | www.plosgenetics.org 11 April 2010 | Volume 6 | Issue 4 | e1000913