Clr-C Required for Heterochromatin Establishment Chp1 occupancy on centromeric dh repeats relative to adh1+, measured by real time PCR. ChIP values were normalized to strains lacking clr4+, and are the mean 6 SEM of two independent ChIP experiments. Strains analyzed as in (A). C. Real time PCR quantitation of ChIP of H3K9me2 at centromeric dh repeats in tas3-TAP and tas3WG-TAP strains following reintegration of clr4+, raf1+, and rik1+ into knockout backgrounds, measured relative to adh1+. Data represents mean 6 SEM of 2 independent ChIP experiments and combine analyses for 2 reintegrants. Data were normalized to a strain lacking clr4D (PY1838). Strains analyzed PY3494, 3497, 1838, 2678, 2679, 2680, 2681, 3776, 4879, 4880, 4881, 4882, 3287, 3707, 3708, 3710, 3711. D. Real time PCR analysis of ChIP material derived from strains analyzed in (C), assessed for Chp1 association with centromeric dh repeats (site A). Data were processed as described in (C). doi:10.1371/journal.pgen.1001174.g004 Figure 5. cid12+ and hrr1+ RDRC components are not required to initiate silencing of centromeric heterochromatin. A. Serial dilution spotting assay to monitor the role of cid12+ in the silencing of the cen::ura4+ reporter gene. Cells of the indicated genotypes and carrying the cen::ura4+ reporter were plated onto selective media. Following reintegration of cid12+ into the cid12D locus (cid12D to cid12+), both tas3-TAP and tas3WG-TAP cells grew on FOA media. Strains analyzed were PY2036, 4338, 4340, 4423, 4424, 4425. B. Serial dilution spotting assay of strains carrying the cen::ura4+ reporter, following hrr1+ reintegration at the hrr1D locus (hrr1D to hrr1+) and assessed for ura4 expression compared with control strains on selective media. Strains analyzed: PY2036, 1838, 4334, 4336, 4420, 4481, 4586, 4587. C. Real time PCR analysis of cDNAs derived from indicated strains, quantifying centromeric dh transcripts relative to adh1+ and normalized to ratio obtained in wild type cells. Data represent mean 6 SEM from 2 independent RNA preparations. Strains analyzed: PY2036,1838, 3494, 3497, 4312, 4423, 4424, 4425. See also Figure S3A. D. Small RNA northern blot was hybridized with a probe for centromeric dh siRNAs, and with a probe for snoR69 as a loading control. Small RNAs were derived from strains used in (C). E. Real time PCR analysis of dh relative to adh1+ transcripts in cDNA derived from indicated strains. Values represent means of 2 RNA preparations 6 SEM. Strains analyzed: PY2036, 1838, 3494, 3497, 4308, 4420, 4481, 4586, 4587. See also Figure S3B. F. Northern blot for small RNA species hybridizing to the cen dh probe and to snoR69 as loading control. Strains are listed in (E). doi:10.1371/journal.pgen.1001174.g005 PLoS Genetics | www.plosgenetics.org 9 October 2010 | Volume 6 | Issue 10 | e1001174