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| Status |
Public on May 22, 2013 |
| Title |
drug_1-10-PHE_ic90 [nucleosome profile] |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
yeast strain BY4741 grown in the presence of 100μM 1,10-Phenanthroline for 2 hours (IC90)
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741, MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
molecule subtype: Nucleosomal DNA from the indicated test strain
|
| Growth protocol |
The haploid yeast strain BY4741 (parental strain of the haploid yeast deletion collection) was used for drug treatments. Cells were grown overnight and then diluted to an OD600 of 0.4. The drugs Nicotinamide (82 mM, IC50), Sodium Butyrate (20 mM, IC50), 1,10-Phenanthroline (100 μM, IC90) or 6-Azauracil (6 mM, IC90), all obtained from Sigma-Aldrich, were added to the media for 2 hours. Drug concentrations were predetermined to inhibit growth by 50% or 90%.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked by direct addition of methanol-free formaldehyde (Polysciences) to a final concentration of 2% for 30 min while shaking at 30 °C. The reaction was quenched by adding glycine to a final concentration of 125 mM for 5 min. Cells were pelleted, washed with 20 mL phosphate buffered saline solution once, and resuspended in 6 mL of [1 M sorbitol, 50 mM Tris 7.4] with freshly added 10 mM β-mercaptoethanol in a 15 mL conical tube. Zymolyase (20T, TakaRa Biotechnology Co., Ltd, Japan) was added to a final concentration of 0.25 mg/mL and cells were spheroplasted at 30 °C while gently rolling for 30 min. After zymolyase treatment, cells were pelleted and resuspended in 4 mL of [1 M sorbitol, 50 mM NaCl, 10 mM Tris 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.075% NP-40] with freshly added 1 mM β-mercaptoethanol and 500 μM spermidine. Spheroplasts were divided into 6 aliquots of 300 μL each and transferred into 1.5 mL Eppendorf tubes. Micrococcal nuclease (MNase; Worthington) dissolved in water at 0.1 U/μL stock was added to the tubes at concentrations of 0, 25, 50, 75, 100, and 150 U per sample. The digestion reactions were incubated at 37 °C for 45 min and reactions were stopped by adding 75 μL of [5% SDS, 50 mM EDTA]. Remaining proteins were digested by adding 3 uL of 20 mg/mL proteinase K solution (Qiagen) was added to each tube for an overnight incubation at 65 °C. DNA from each MNase aliquot was isolated by phenol/chloroform extraction, concentrated via ethanol precipitation and treated with RNaseA (Fermentas) at a final concentration of 1mg/ml for 1 hour. Samples were then separated on a 2% agarose gel and bands corresponding to ~147bp (mono-nucleosomal fragments) were gel-extracted using the QIAquick kit (Qiagen) for each of the digestions.
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| Label |
Biotin
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| Label protocol |
Mono-nucleosomal DNA was fragmented by nuclease digestion in a solution containing 1X One-Phor-All buffer (GE Healthcare) and DNase I (GE Healthcare) at 37C until the average fragment size was approximately 70 bp. The fragmentation reaction was stopped by incubating at 95C for 10 min. Fragmented samples were biotinylated with Biotin-N6-ddATP (Enzo Life Sciences) using terminal deoxynucleotidyl transferase at 37C for 2 hours.
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| Channel 2 |
| Source name |
MNase-treated total genomic DNA from WT (BY4741) strain
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741, MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
|
| Growth protocol |
The haploid yeast strain BY4741 (parental strain of the haploid yeast deletion collection) was used for drug treatments. Cells were grown overnight and then diluted to an OD600 of 0.4. The drugs Nicotinamide (82 mM, IC50), Sodium Butyrate (20 mM, IC50), 1,10-Phenanthroline (100 μM, IC90) or 6-Azauracil (6 mM, IC90), all obtained from Sigma-Aldrich, were added to the media for 2 hours. Drug concentrations were predetermined to inhibit growth by 50% or 90%.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked by direct addition of methanol-free formaldehyde (Polysciences) to a final concentration of 2% for 30 min while shaking at 30 °C. The reaction was quenched by adding glycine to a final concentration of 125 mM for 5 min. Cells were pelleted, washed with 20 mL phosphate buffered saline solution once, and resuspended in 6 mL of [1 M sorbitol, 50 mM Tris 7.4] with freshly added 10 mM β-mercaptoethanol in a 15 mL conical tube. Zymolyase (20T, TakaRa Biotechnology Co., Ltd, Japan) was added to a final concentration of 0.25 mg/mL and cells were spheroplasted at 30 °C while gently rolling for 30 min. After zymolyase treatment, cells were pelleted and resuspended in 4 mL of [1 M sorbitol, 50 mM NaCl, 10 mM Tris 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.075% NP-40] with freshly added 1 mM β-mercaptoethanol and 500 μM spermidine. Spheroplasts were divided into 6 aliquots of 300 μL each and transferred into 1.5 mL Eppendorf tubes. Micrococcal nuclease (MNase; Worthington) dissolved in water at 0.1 U/μL stock was added to the tubes at concentrations of 0, 25, 50, 75, 100, and 150 U per sample. The digestion reactions were incubated at 37 °C for 45 min and reactions were stopped by adding 75 μL of [5% SDS, 50 mM EDTA]. Remaining proteins were digested by adding 3 uL of 20 mg/mL proteinase K solution (Qiagen) was added to each tube for an overnight incubation at 65 °C. DNA from each MNase aliquot was isolated by phenol/chloroform extraction, concentrated via ethanol precipitation and treated with RNaseA (Fermentas) at a final concentration of 1mg/ml for 1 hour. Samples were then separated on a 2% agarose gel and bands corresponding to ~147bp (mono-nucleosomal fragments) were gel-extracted using the QIAquick kit (Qiagen) for each of the digestions.
|
| Label |
Biotin
|
| Label protocol |
Mono-nucleosomal DNA was fragmented by nuclease digestion in a solution containing 1X One-Phor-All buffer (GE Healthcare) and DNase I (GE Healthcare) at 37C until the average fragment size was approximately 70 bp. The fragmentation reaction was stopped by incubating at 95C for 10 min. Fragmented samples were biotinylated with Biotin-N6-ddATP (Enzo Life Sciences) using terminal deoxynucleotidyl transferase at 37C for 2 hours.
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| Hybridization protocol |
6 μg of biotinylated cDNA was hybridized at 45C for 16 hours, and then washed and stained according to Affymetrix protocol EukGE-WS2v4_450 in an Affymetrix Fluidics Station 450.
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| Scan protocol |
Arrays were scanned in an Affymetrix 7G scanner.
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| Description |
ctrl CEL files:
gDNA-control_rep1.CEL; gDNA-control_rep2.CEL;
gDNA-control_rep3.CEL; gDNA-control_rep4.CEL
Monococcal nuclease treated chromatin profile for yeast strain BY4741 grown in the presence of 100μM 1,10-Phenanthroline for 2 hours (IC90) compared to genomic DNA control
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| Data processing |
Raw data files were normalized with Affymetrix Tiling Analysis Software (TAS) v1.1. Treatment and control samples were quantile normalized in pairs using only perfect match probes with a bandwidth of 20.
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| Submission date |
Mar 05, 2013 |
| Last update date |
May 22, 2013 |
| Contact name |
Harm van Bakel |
| E-mail(s) |
[email protected]
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| Organization name |
Mount Sinai School of Medicine
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| Department |
Genetics and Genomic Sciences
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| Lab |
Bakel Lab
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| Street address |
One Gustave L. Levy Place, Box 1498
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| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10029 |
| Country |
USA |
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| Platform ID |
GPL7070 |
| Series (2) |
| GSE44877 |
A compendium of nucleosome and transcript profiles reveals determinants of chromatin architecture and transcription [nucleosome profiling] |
| GSE44879 |
A compendium of nucleosome and transcript profiles reveals determinants of chromatin architecture and transcription |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM1093312_drug_1-10-PHE_ic90.CEL.gz |
31.5 Mb |
(ftp)(http) |
CEL |
| GSM1093312_drug_1-10-PHE_ic90.bar.gz |
18.7 Mb |
(ftp)(http) |
BAR |
| Processed data provided as supplementary file |
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