the intriguing possibility that, similar to other histone modifying enzymes such as the Set1 and Set2 methyltransferases, that Clr-C may associate with, and be brought to chromatin, via RNA polymerase II [39]. Another mutation in RNA pol II that causes defective heterochromatin assembly resides in the Rpb7 subunit [16], which together with its partner, Rpb4, is thought to be an accessory and non-obligate component of yeast RNA pol II. One interesting possibility would be if Clr-C recruitment to regions of chromatin that are destined to become heterochromatic was controlled by modulation of RNA pol II by the Rpb4/7 subcomplex. In contrast, in plants, two pol II-related RNA polymerase activities, Pol IV and Pol V, have evolved to mediate heterochromatin assembly on repetitive sequences (reviewed in [40]). Materials and Methods Plasmid construction Integration plasmids for genomic clones were constructed by PCR using Phusion polymerase (NEB) and standard cloning or Gateway (Invitrogen) techniques. Oligonucleotide sequences are listed in Table S2. Full details of plasmid construction are listed in Text S1. Strain generation Strains used in this study are listed in Table S1 and details of their construction and verification are in Text S1. Serial dilution assays on selective media Cells were cultured overnight at 25uC in rich YES medium to a density of approximately 56106 cells/ml. Cells were washed extensively in PMG media, counted, and five-fold serial dilutions made, such that plating of 4 ul of cells yielded 1.26104 cells within the most concentrated spot. Plating was performed on PMG complete media, PMG media lacking uracil, and PMG complete media supplemented with 2g FOA per liter as described previously [21], and incubated for 5 days at 25uC. Transcript and siRNA analyses Transcript and siRNA analyses were performed as previously described [10,21]. Oligos for real time PCR analysis: (dh) JPO-769 and JPO-770, (dg) JPO-986, JPO-987, adh1, JPO-793 and JPO-794 [10]. RNA was prepared from duplicate cultures for every experiment, and for analyses following gene reintegration, multiple independent re-integrants were assessed. Real-time PCR was performed on an Eppendorf Mastercycler ep Realplex machine using Quantifast Sybr green (Qiagen). Data was analyzed using the DCt method, ensuring that all samples gave Ct values within the experimentally determined linear range. Chromatin Immunoprecipitation analyses Chromatin immunoprecipitation was performed as previously described [10,21], using antibodies that recognize H3K9me2 (Abcam) and Chp1 (Abcam). Further details are in Text S1. Supporting Information Figure S1 Analysis of transcript accumulation from dg sites within the centromere by real time PCR analysis. A. Transcripts from cen dg sequences were measured by real time PCR relative to adh1+ transcript accumulation in cDNA derived from strains listed in Figure 2C (raf1D to raf1+). Duplicate RNA preparations were used to generate duplicate cDNAs and data represent mean 6 SEM. B. Centromeric transcripts from the dg region of the Clr-C Required for Heterochromatin Establishment centromere were measured by real time PCR of cDNA derived from strains listed in Figure 2E (raf2D to raf2+). Analysis was performed as described above. Found at: doi:10.1371/journal.pgen.1001174.s001 (0.01 MB PDF) Figure S2 Analysis of strains bearing genomic reintegration of dcr1+. A. Serial dilution assay to monitor growth of cen::ura4+ reporter strains on non-selective media (complete), media lacking uracil (2URA), or media supplemented with FOA (+FOA). Following reintegration of dcr1+ into the genomic dcr1D locus, both tas3-TAP and tas3WG-TAP isolates were able to grow on FOA. Strains used were PY2036, 3310, 3307, 3501, 3502, 3499, 3500. B. Real time PCR analysis of centromeric transcript accumulation from the dh repeat sequences relative to adh1+ transcript accumulation in cDNA derived from the indicated strains. Two independent reintegrants of dcr1+ (dcr1D to dcr1+) were analyzed for both the tas3-TAP and tas3WG-TAP backgrounds. Data represent mean 6 SEM for cDNA samples from 2 independent RNA preparations for each strain. Data was normalized to wild type cen::ura4+ strain (PY2036), which was set at 1. Strains used were PY2036, 3310, 3307, 3501, 3502, 3499, 3500. C. Centromeric dg transcripts were measured by similar methods and using the same cDNA samples as used for (B). D. Northern blotting for small RNA species in RNA preparations from the strains listed in (B). Blot was probed for siRNAs derived from dh repeats and for the snoR69 RNA as a loading control. Found at: doi:10.1371/journal.pgen.1001174.s002 (0.24 MB PDF) Figure S3 Analysis of centromeric dg and dh transcript accumulation by real time PCR. A. Real time PCR analysis of dg centromeric transcripts relative to adh1+ in cDNA derived from strains listed in Figure 5C (cid12D to cid12+). Duplicate RNA preparations were used to generate duplicate cDNAs and data represent mean 6 SEM. B. Real time PCR analysis of transcripts derived from dg centromeric repeats relative to adh1+ in strains listed in Figure 5E (hrr1D to hrr1+). Analysis was performed as described above. Found at: doi:10.1371/journal.pgen.1001174.s003 (0.02 MB PDF) Figure S4 Analysis of centromeric transcript accumulation in rdp1 cells and Chp1 association with centromeric dh sequences in hrr1 mutant cells. A. Real time PCR analysis of transcripts derived from dh centromeric repeats relative to adh1+ in strains listed in Figure 6C (rdp1D to rdp1+). Analysis was performed as described in Figure 6C. B. ChIP was performed with Chp1 antibodies on chromatin prepared from strains listed in Figure 6D. Real time PCR was used to quantify Chp1 association with dh relative to the euchromatic adh1 control. Data was normalized to cells lacking clr4 (set at 1), in which Chp1 does not associate with centromeres. Error bars represent the SEM of duplicate ChIPs using different biological samples. Found at: doi:10.1371/journal.pgen.1001174.s004 (0.02 MB PDF) Figure S5 Transient depletion of ago1+ does not affect establishment of silencing of centromeric transcripts in tas3WG cells. A. Real Time PCR analysis of cDNA prepared from indicated strains, measuring centromeric dg transcript accumula- tion normalized to adh1+ expression. Data represents mean average of analysis of 2 independent cDNA preparations from duplicate biological samples, measuring two independent ago1+ reintegrants for each background, with error bars representing SEM. Strains used were as in Figure 7A. Found at: doi:10.1371/journal.pgen.1001174.s005 (0.01 MB PDF) Table S1 Strains used in this study. Found at: doi:10.1371/journal.pgen.1001174.s006 (0.11 DOC) MB PLoS Genetics | www.plosgenetics.org 15 October 2010 | Volume 6 | Issue 10 | e1001174