Fig. 7. Telomeric pull-down assay. Telomeric insertions of uninsulated (lines 801 and 856) and insulated (lines 605, 613, and 615) transgenes and random integrants (lines 1001 and 1007) are shown. As in the Fig. 2, genomic DNA integration of telomeric and random clones was evaluated by a digestion with the enzyme DraIII, which releases the transgene along with the telomeric repeats. Digested DNA was annealed to telomeric-specific biotinylated oligonucleotides, and telomere-oligonucleotide complexes were captured with streptavidin-coated magnetic beads. The unbound and bound DNA fractions were resolved on agarose gel and probed with a ³²P-labeled GFP probe. Notice the presence of the smear on the 600 and 800 series and the absence of hybridization signal on the 1000 series.

Fig. 8. ChIP assay of the 801 and 856 uninsulated and telomeric clones, integrated in telomeric regions (Fig. 2 and SI Fig. 7). These lines were rapidly silenced (see Fig. 3A) and present a chromatin conformation enriched on repressive marks. Relative enrichment of histone modification was plotted, and average enrichment values of at least two independent immunoprecipitations are shown. Each PCR was performed at least twice. The upper band corresponds to the GFP amplification product, and the lower band corresponds to the product from control primers used for normalization.

Fig. 9. ChIP-re-ChIP assay. The re-ChIP experiment was performed essentially as in refs. 1 and 2. H3K4me2 or H3K9me3 antibodies were used for the first round of immunoprecipitation (IP), and the order was reversed for the second round of IP. As control of the ChIP each antibody was used in a single round of IP. The H3K4me2 and H3K9me3 individual ChIP assays were normalized against the input. The ChIP-re-ChIP experiments were normalized against an IgG for the first round of IP and the corresponding histone mark antibody for the second round of IP. We show a representative duplex PCR performed in triplicate, quantitated and plotted with the standard error of one of the two independent ChIP-re-ChIP experiments performed. The upper band corresponds to the GFP amplification product, and the lower band corresponds to the product from control primers used for normalization.

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