treatment_ChIP-chip: ChIP was carried out as previously described (van Bakel et al., 2008) with some modifications. Cells were spheroplasted according to the protocol of the Rando lab (Rando, 2010) and then directly sonicated (Bioruptor, Diagenode: ten cycles, 30 sec on/off, medium setting). 200 µL chromatin extract was incubated with 10 µL of anti-GFP antiserum (homemade) (3h, RT) which had been coupled to Protein G agarose beads (Roche 11 243 233 001) overnight at 4oC. After incubation, antibody ChIPs were washed twice in FA lysis buffer (50 mM HEPES KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS), twice with FA lysis buffer containing 0.5 M NaCl, and twice with 10 mM Tris at pH 8.0, 0.25 mM LiCl, 1 mM EDTA, 0.5% Nonidet P-40 and 0.5% Na-deoxycholate. Cross-links of the ChIP samples were reversed overnight at 65°C in 150 μL 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% SDS.
Growth protocol
growth_ChIP-chip: O/N cultures of WT-aa and Hsf1-aa were diluted in synthetic complete (SC) medium containing 2% glucose and grown for two doublings. Subsequently, a low dose of rapamycin (0.15 µM) or the same volume of DMSO (t0) was added to the cells for the indicated amount of time. Additions were staggered so that all the cultures could be crosslinked at the same time and OD (90 minutes after the first additions: 3 doublings). Crosslinking was done with a final concentration of 2% formaldehyde for 30 minutes at 30oC. Crosslinking was quenched with glycine (final concentration = 125 mM) for 5 minutes. The cells were harvested by centrifugation, washed once in MilliQ and subsequently snap frozen in liquid nitrogen.
Extracted molecule
genomic DNA
Extraction protocol
ChIP-DNAextraction: Reverse crosslinked DNA samples were incubated with 400 µg proteinase K (Roche) for 2 hours at 37°C. For ChIP-chip, the proteinase K step was preceded by shrimp alkaline phosphatase (SAP) treatment by adding 1 ul of SAP (Roche) for 2 hours at 37°C. DNA was extracted with phenol-chloroform-isoamylalcohol (Sigma), separated using Phaselock tubes and cleaned on PCR purification columns (Qiagen).
Label
Cy5
Label protocol
ChIP-amplification: Input and ChIP DNA was amplified using a robotically automated double-round T7 RNA polymerase-based amplification procedure [Van Bakel H, van Werven FJ, Radonjic M, Brok MO, van Leenen D, et al. (2008) Improved genome-wide localization by ChIP-chip using double-round T7 RNA polymerase-based amplification. Nucleic Acids Res 36]. ChIP samples were hybridised with input DNA to a high-resolution 44K yeast array (Agilent Technologies).
treatment_ChIP-chip: ChIP was carried out as previously described (van Bakel et al., 2008) with some modifications. Cells were spheroplasted according to the protocol of the Rando lab (Rando, 2010) and then directly sonicated (Bioruptor, Diagenode: ten cycles, 30 sec on/off, medium setting). 200 µL chromatin extract was incubated with 10 µL of anti-GFP antiserum (homemade) (3h, RT) which had been coupled to Protein G agarose beads (Roche 11 243 233 001) overnight at 4oC. After incubation, antibody ChIPs were washed twice in FA lysis buffer (50 mM HEPES KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS), twice with FA lysis buffer containing 0.5 M NaCl, and twice with 10 mM Tris at pH 8.0, 0.25 mM LiCl, 1 mM EDTA, 0.5% Nonidet P-40 and 0.5% Na-deoxycholate. Cross-links of the ChIP samples were reversed overnight at 65°C in 150 μL 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% SDS.
Growth protocol
growth_ChIP-chip: O/N cultures of WT-aa and Hsf1-aa were diluted in synthetic complete (SC) medium containing 2% glucose and grown for two doublings. Subsequently, a low dose of rapamycin (0.15 µM) or the same volume of DMSO (t0) was added to the cells for the indicated amount of time. Additions were staggered so that all the cultures could be crosslinked at the same time and OD (90 minutes after the first additions: 3 doublings). Crosslinking was done with a final concentration of 2% formaldehyde for 30 minutes at 30oC. Crosslinking was quenched with glycine (final concentration = 125 mM) for 5 minutes. The cells were harvested by centrifugation, washed once in MilliQ and subsequently snap frozen in liquid nitrogen.
Extracted molecule
genomic DNA
Extraction protocol
ChIP-DNAextraction: Reverse crosslinked DNA samples were incubated with 400 µg proteinase K (Roche) for 2 hours at 37°C. For ChIP-chip, the proteinase K step was preceded by shrimp alkaline phosphatase (SAP) treatment by adding 1 ul of SAP (Roche) for 2 hours at 37°C. DNA was extracted with phenol-chloroform-isoamylalcohol (Sigma), separated using Phaselock tubes and cleaned on PCR purification columns (Qiagen).
Label
Cy3
Label protocol
ChIP-amplification: Input and ChIP DNA was amplified using a robotically automated double-round T7 RNA polymerase-based amplification procedure [Van Bakel H, van Werven FJ, Radonjic M, Brok MO, van Leenen D, et al. (2008) Improved genome-wide localization by ChIP-chip using double-round T7 RNA polymerase-based amplification. Nucleic Acids Res 36]. ChIP samples were hybridised with input DNA to a high-resolution 44K yeast array (Agilent Technologies).
Hybridization protocol
Tecan HS4800 hybridization: * 60 ul labeled sample is combined with 60 ul 2x-hybmix, containing 50% formamide, 10xSSC, 0.2% SDS, 200 ug/ml herring sperm DNA * Hybridizations of spotted oligo-arrays (Codelink glass) or Agilent microarrays are performed on a HS4800Pro Hybstation (Tecan) * Priming: 5xSSC, 0.1%SDS * Probe injection: pre-hyb, 5xSSC, 25% formamide, 0.1%SDS, 1%BSA, Volume 110ul * Hybridization: 45 min at 42C. * Wash 2x: milliQ * Wash: 5xSSC, 0.1%SDS * Probe injection: sample. Volume 110ul (Agilent 4packs: 55ul) * Hybridization: 16 hours at 42C. * Wash 2x: 1xSSC, 0.2%SDS at 23C * Wash 2x: 0.1xSSC, 0.2%SDS at 23C * Wash 2x: 0.1xSSC at 23C * Drying: blow with nitrogen for 3min at 30C
Scan protocol
Scanning of slides using the Agilent G2565BA scanner. Features were extracted using ImaGene software from Biodiscovery.
Data processing
Genes nobg, hybset spec. dye, density: Combination of lowess normalization using genes no backgroundcorrection, hybset specific gene specific dye bias correction and density selection. limma: A software package for the analysis of gene expression microarray data, especially the use of linear models for analysing designed experiments and the assessment of differential expression. Author(s): Gordon Smyth. The limma R package version 2.12.0 is used. P-values are Benjamini-Hochberg FDR corrected.
