The proposed normalization procedure: (a) The MA plot before normalization shows a need for rotation to correct dye-bias

<p><b>Copyright information:</b></p><p>Taken from "Normalization and experimental design for ChIP-chip data"</p><p>http://www.biomedcentral.com/1471-2105/8/219</p><p>BMC Bioinformatics 2007;8():219-219.</p><p>Published online 25 Jun 2007</p><p>PMCID:PMC1913544.</p><p></p> (b) To determine the correct angle of rotation, the () vs () plot of the differences between probes is generated (the differences were taken between probes that are 800 bp along the chromosome; see Figure 8). This circumvents the effect of binding signal in determining the rotating angle for original MA plot in (a). (c) The MA plot after rotation by the angle determined in (b). The green line is the lowess fitting line after rotation. (d) The MA plot after lowess normalization. 3000 sample points from each of X and 2L chromosomes are shown

The Scatter plot of ChIP-chip versus mock control after rotation and lowess normalization (cf

<p><b>Copyright information:</b></p><p>Taken from "Normalization and experimental design for ChIP-chip data"</p><p>http://www.biomedcentral.com/1471-2105/8/219</p><p>BMC Bioinformatics 2007;8():219-219.</p><p>Published online 25 Jun 2007</p><p>PMCID:PMC1913544.</p><p></p> Figure 3), (a) without and (b) with running median smoothing. The optimal line for separating the X-specific signals from the 2L signals is now a horizontal line. This suggests that mock control data correction may be neglected when a proper normalization is carried out

Additional file 1: Figure S1. of Comprehensive analysis of promoter-proximal RNA polymerase II pausing across mammalian cell types

Measurement of pausing index (PI). Figure S2. Robustness of RNAP2 pausing calculations. Figure S3. Pausing across cell and tissue types. Figure S4. Correlation between pausing and promoter GC and CpG content Figure S5. Relationship between whole gene, TSSR, and gene body RNAP2 density and PI to gene expression for GM12878, H1, K562, IMR90, HUVEC, and HepG2 cells. Figure S6. Grouping paused and non-paused genes by mean population-wide expression shows no consistent expression level difference within quantile. Figure S7. Effect of extracellular stimuli on RNAP2 pausing. Figure S8. Relationship of gene expression to TSSR and gene body RNAP2 density and to PI. Figure S9. The inflection point does not appear to be driven by a limit to the level of initiating RNAP2. Figure S10. Nucleosome positioning and RNAP2 pausing. Figure S11. Chromatin features and RNAP2 pausing. (PDF 7287 kb

Additional file 3: Table S2. of Comprehensive analysis of promoter-proximal RNA polymerase II pausing across mammalian cell types

GO biological process enrichment analysis for the top and bottom 25 % of genes by PI for each cell type. (XLSX 23028 kb

Additional file 2: Table S1. of Comprehensive analysis of promoter-proximal RNA polymerase II pausing across mammalian cell types

Datasets used in this study. (XLSX 19 kb

Additional file 4: Table S3. of Comprehensive analysis of promoter-proximal RNA polymerase II pausing across mammalian cell types

List of primers used in this study. (XLSX 10 kb

CLAMP and MSL complex promote accessibility at CES.

<p>A) MACC values at Chromatin Entry Sites (CES) in male (S2) cells are greatest at the peak center (control RNAi, blue). RNAi treatment of <i>clamp</i> reduces accessibility at the CES peak and +/- 500 bp beyond the peak (green). For all panels, the lighter color indicates 95% confidence intervals, while the darker line represents the average MACC value. B.) A reduction in MSL complex following <i>msl2</i> RNAi only reduces accessibility directly at the CES peak (purple). C) The average MACC scores around CES in female Kc cells indicates a reduction in MACC values distal to the CES center, extending +/- 500bp beyond the peak. D) MACC scores at the three subgroups of CES indicate <i>clamp</i> RNAi (green) results in a decrease in accessibility in CES Groups A and B, but not in Group C. E) RNAi targeting <i>msl2</i> (purple) results in a loss of accessibility at Group A CES only at the CES peak. There is a small reduction in accessibility in Group B sites and no effect of <i>msl2</i> RNAi treatment at Group C sites. F) MACC scores from Kc cells are plotted for the three subgroups of CES. In females, <i>clamp</i> RNAi (green) results in a decrease in MACC values around CES in Group A, and to a lesser extent Group B. CES in Group C exhibit an increase in MACC values following <i>clamp</i> RNAi.</p

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CLAMP directly affects accessibility of X-linked gene bodies in both males and females.

<p>A) The difference in MACC value (RNAi treatment—control) over gene bodies of all annotated genes was ranked by the magnitude of decrease (top) to increase (bottom) in accessibility. RNAi of <i>msl2</i> shows only a modest change in accessibility, whereas <i>clamp</i> RNAi results in accessibility changes in both males and females. Shown to the right of each heat map is the percent of genes that decrease (MACC<0, blue line) or increase (MACC>0, red line) in accessibility following the indicated RNAi treatment. B) Average MACC profiles across gene bodies are shown for male (S2) and female (Kc) cells separated by X-chromosomes and autosomes. X-linked gene bodies in both males and females have reduced accessibility after <i>clamp</i> RNAi treatment (green). The dark line represents the average MACC value, while 95% confidence intervals are represented by the lighter shading. C) The difference in MACC scores (Δ MACC) between control and RNAi over gene bodies was calculated. Plotted is the distribution of difference values for all chromosomes together (all), or separately for the X-chromosome and autosomes. Plotted is the median difference in MACC scores with the 95% confidence interval indicated by a notch around the median line.</p

CLAMP promotes nucleosome positioning at 5’ends genome-wide.

<p>A) Average MACC profiles around transcription start sites (TSS) are shown for male (S2) and female (Kc) cells separated by X-chromosomes and autosomes. TSS at genes with highly bound CLAMP in both males and females are reduced in accessibility after <i>clamp</i> RNAi treatment (green) compared to control (blue). The dark line represents the average MACC value, while 95% confidence intervals are represented by the lighter colors. B) Average MNase-seq read frequency was calculated for the four MNase experiments and plotted +/- 1 Kb flanking annotated TSS. Upon <i>clamp</i> RNAi (green), there is a decrease in read frequency upstream of the TSS with a concurrent increase in occupancy within gene bodies.</p