Clr-C Required for Heterochromatin Establishment Figure 6. RDRC components are not required for establishment of silent H3K9me2 chromatin at centromeres. A. Serial dilution assay of strains bearing the cen::ura4+ reporter and of the indicated genotype, plated on selective media. Strains analyzed: PY2036, 4300, 4304, 4401, 4402, 4403 and 4404. B. Northern blotting for small RNAs derived from dh centromeric repeats, with snoR69 RNA as loading control. Strains analyzed: PY 2036, PY1838, 3494, 3497, 4274, 4401, 4402, 4403, 4404. C. Real time PCR analysis of cDNA derived from strains of the indicated genotypes (as for Figure 6B), measuring centromeric dh transcripts normalized to adh1+, and to a wild type strain (PY2036). Data represents the mean 6 SEM from analysis of 2 independent cDNA preparations from duplicate biological samples. See also Figure S4A. D. Real time PCR analysis of ChIP material derived using antibodies against H3K9me2, analyzed for centromeric dh sequences and normalized to euchromatic adh1+ sequences. Data represent mean from two independent experiments 6 SEM, normalized to data obtained from clr4D cells. Strains analyzed: PY2036, 1838, 3494, 3497, 4308, 4420, 4481, 4586, 4587. See also Figure S4B. E. ChIP for H3K9me2 at centromeric dh sequences relative to adh1+, normalized to a clr4D strain. Duplicate ChiP experiments were performed and data were processed as described above. Strains analyzed included dcr1+ reintegration strains (dcr1D to dcr1+), rdp1+ reintegration strains (rdp1D to rdp1+) and cid12+ reintegrants (cid12D to cid12+). Two independent gene reintegrants for each tas3WG-TAP strain background were tested in duplicate. Strains analyzed: PY 2036, 3494, 3497, 3235, 3501, 3502, 3499, 3500, 4274, 4401, 4402, 4403, 4404, 4312, 4423, 4424, 4425. doi:10.1371/journal.pgen.1001174.g006 PLoS Genetics | www.plosgenetics.org 10 October 2010 | Volume 6 | Issue 10 | e1001174