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Sample GSM2179675 Query DataSets for GSM2179675
Status Public on Nov 11, 2016
Title HSF1_t60_slow_4_IP vs. HSF1_t60_slow_4_INPUT
Sample type genomic
 
Channel 1
Source name HSF1_t60_slow_IP
Organism Saccharomyces cerevisiae
Characteristics strain/background: S288C-BY4742
genotype/variation: Hsf1-aa : tor1-1; fpr1del; RPL13A-FKBP12-NAT; Hsf1-FRB-yEGFP
treatment: HSF1_t60_slow
chip antibody: anti-GFP
Treatment protocol 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).
 
Channel 2
Source name HSF1_t60_slow_INPUT
Organism Saccharomyces cerevisiae
Characteristics strain/background: S288C-BY4742
genotype/variation: Hsf1-aa : tor1-1; fpr1del; RPL13A-FKBP12-NAT; Hsf1-FRB-yEGFP
treatment: HSF1_t60_slow
chip antibody: none
Treatment protocol 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.
 
Submission date May 27, 2016
Last update date Nov 11, 2016
Contact name Marian Groot Koerkamp
Organization name Princess Maxima Center for Pediatric Oncology
Department Research
Lab Drostlab
Street address Heidelberglaan 25
City Utrecht
State/province Utrecht
ZIP/Postal code 3584 CS
Country Netherlands
 
Platform ID GPL21864
Series (2)
GSE81481 HSF1 and MOT1-expression and binding: Molecular mechanisms that distinguish TFIID housekeeping from regulatable SAGA promoters
GSE81987 Hsf1-ChIP-on-chip: Molecular mechanisms that distinguish TFIID housekeeping from regulatable SAGA promoters

Data table header descriptions
ID_REF
VALUE Normalized log2 ratio (Cy5/Cy3)
Signal Norm_Cy5 Normalized Cy5 signal intensity
Signal Norm_Cy3 Normalized Cy3 signal intensity

Data table
ID_REF VALUE Signal Norm_Cy5 Signal Norm_Cy3
1 0.404882907 89.8663 67.8758
2 0.5577195 100.035 67.9613
3 0.145263531 74.344 67.2229
4 0.66902131 125.337 78.8286
5 0.233892167 74.7523 63.5647
6 0.422989329 79.9642 59.6435
7 0.334776014 76.1818 60.4051
8 0.11789146 71.3107 65.7152
9 0.192545689 75.8934 66.4113
10 0.296011631 80.6317 65.6746
11 0.311562062 80.2101 64.6308
12 0.151643856 75.5804 68.0393
13 0.384313846 85.4968 65.5028
14 0.343911745 81.0404 63.8519
15 0.507331834 106.321 74.7992
16 -0.723550235 983.176 1623.46
17 0.064835413 159.174 152.179
18 1.045188152 316.572 153.405
19 -0.093259033 365.768 390.193
20 -0.169031851 106.642 119.898

Total number of rows: 45220

Table truncated, full table size 1496 Kbytes.




Supplementary file Size Download File type/resource
GSM2179675_13441_raw.txt.gz 1.9 Mb (ftp)(http) TXT
Processed data included within Sample table
Processed data are available on Series record

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