were endogenously tagged as previously described [39]. For ChIP analysis cells were grown in 50 mL of YPD to an OD600 of 0.6–0.8 before crosslinking with 1% formaldehyde for 30 min. Chromatin Immunoprecipitation and Antibodies ChIP experiments were performed as per [52], with minor modifications. For myc-tag ChIP, we used 5 mg of 9E11 antibody coupled to 26107 pan-mouse IgG DynaBeads (Invitrogen) per sample. RNAPII ChIPs were done using 2 mL of 8WG16 antibody coupled to 26107 panmouse IgG DynaBeads per sample. For Ser2-P and Ser5-P ChIP, we used 100 mL of rat serum (3E8 and 3E10 respectively) coupled to 26107 protein G DynaBeads per sample [53]. The microarrays used for location analysis were purchased from Agilent Technologies (Palo Alto, California, United States) and contain a total of 44,290 Tm-adjusted 60-mer probes covering the entire genome for an average density of one probe every 275 bp (6100 bp) within the probed regions (4X44K). Myc-tag ChIPs were hybridized against ChIPs from isogenic strains that did not contain the tag as controls. RNAPII ChIPs were hybridized against a sample derived from 400 ng of input (non-immunopre- cipitated) DNA. Quantitative real-time PCR analysis was performed using the ABI 7000 machine (Applied biosciences) and Sybr green PCR mastermix (Applied biosystems). The % IP ORF/% IP no ORF ratio was determined using the relative efficiency method and calculated as in Lloyd et al. [54]. Primers were designed using the primer express software (Applied biosciences) exclusively matching the ORF of the indicated genes (listed in table S1). Data Analysis The data was normalized and replicates were combined using a weighted average method as described previously [52]. The log 2 ratio of each spot of combined datasets was then converted to Z- score, similar to Hogan et al. [55] to circumvent the large differences in the immunoprecipitation efficiencies of the different factors. Visual inspection of the Z-scores was carried out on the UCSC Genome Browser (http://genome.ucsc.edu/). All data analyses described here were done using data from protein-coding genes longer than 500 bp. Median Z-score values for promoter and gene coding sequences were calculated and used in our clustering analyses. Promoters are defined as the shortest of either 250 bp or half the intergenic region (half-IG) relative to the reference gene’s 59 boundary. Self-organizing map (SOM) clustering was done with the Cluster software [33] and visualized with Java Treeview [56]. Only genes with no missing value were used for clustering. Gene mapping was performed as in Rufiange et al. [57] on selected groups of genes described in the text. Briefly, data were mapped onto the 59 and 39 boundaries in 50 bp windows for each half-gene and adjacent half- IG regions. A sliding window of 300 bp was then applied to the Z- scores to smooth the curve. GO analyses on clusters were performed with funcassociate 2.0. For this, an association file for the entire gene set was generated and used for all analyses [34]. Regression analysis was plotted using excel, where the x-axis and y-axis contained the whole data set of the average ORF enrichments of the indicated ChIP. A trend line was plotted and the R2 was calculated. Phenotype analysis of rrd1D mutants The H2O2 survival curves were performed as described previously [58], briefly exponentially growing cells were washed once in 50 mM KPO4 (p H7.5) and treated with the indicated concentration of H2O2 (Bio basic Inc) in 50 mM KPO4 (pH 7.5) Rrd1 Regulates Elongation of RNA Polymerase II buffer for one hour. Cells were then scored for colony formation after three days at 30 C on YPD agar. Spottest with NaAs and 6-azauracil (Sigma) analysis was performed as described previously [59]. For 6-azauracil strains were transformed with and empty vector bearing the URA3 gene (pTW423) and spotted onto synthetic media agar plates lacking uracil. For lacZ expression the plasmid bearing the ACR3-LacZ fusion was transformed into strains and exponentially growing cells were treated with 1 mM NaAs (Sigma), aliquots were taken and the b-gal assay was performed as described in [39,60]. mRNA extraction and GeXP analysis Overnight cultures were sub-cultured and grown to an OD600 of 0.6–0.8. Cells were then treated with the indicated drugs for 30 minutes (0.6 mM H2O2, 1 mM NaAs and 100 ng/ml rapamycin). For heat shock, cells were spun down and resuspended in preheated media at 37uC for 30 min. mRNA extraction was performed with the RNeasy extraction kit (Qiagen), following user guide lines. Multiplex analysis by GeXP system Primer design of the set of 10 genes (see suppl. Table S1) was done using the eXpress Designer module of the express Profiler software (Beckman Coulter). 25 ng RNA were used in a 20 mL reaction volume for RT. Kanamycin RNA, an internal positive control was included. The RT reaction was performed using the GenomeLab TM GeXP Start Kit (Beckman Coulter) under the conditions: 1 min at 48uC, 60 min at 42uC, 5 min at 95uC, hold at 4uC, in a thermal cycler. After the RT, a PCR was performed under the conditions: 10 min at 95uC, followed by 35 cycles of 30 s at 94uC, 30 s at 55uC, and 1 min at 70uC. Expression analysis was carried out with the GenomeLab TM GeXP system (Beckman- Coulter) following manufacturer’s instructions using the fragment analysis method. The data was normalized to Kanamycin before being expressed as a log 2 ratio. Statistical analysis For Q-PCR, survival curves and lacZ expression analysis at least three independent experiments were performed and the standard deviation and P-values were calculated (Student’s T-test). For GeXP analysis the percentage CV was calculated for each replicate. Only % CV #20% was taken for analysis. Values.2 SD were excluded before final calculation. Supporting Information Table S1 analysis. (TIF) File S1 (XLS) A list of all primers used for the GeXP and Q-PCR GO analysis of figures 2A. Figure S1 (A) A complement to Figure 2C, same experiment besides that the data was obtained from rapamycin treated cells. (B) A complement of Fig. 2D, the same experiment besides that the data was obtained from rapamycin treated cells. (TIF) Figure S2 (A) Contains the mapping analysis of Ser5-P and Ser2-P of rapamycin unaffected genes (cluster W2 and W5). (B) Mapping of Ser5-P and Ser2-P on clusters S1–S4 from figure 4B. (TIF) Figure S3 Comparison of GeXP mRNA expression and RNAPII median average ChIP data of the corresponding gene PLoS ONE | www.plosone.org 12 August 2011 | Volume 6 | Issue 8 | e23159