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Status |
Public on May 22, 2013 |
Title |
H4-depletion_180min [nucleosome profile] |
Sample type |
genomic |
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Channel 1 |
Source name |
UKY403 after 3 hours of histone H4 depletion
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Organism |
Saccharomyces cerevisiae |
Characteristics |
strain: UKY403, MATa ade2-101 his3-A200 leu2-3, 112 lys-801 trpl-A901 ura3-52 GAL+ thr tyr arg4-1 Ah4-J[HIS3+] Ah4-2[LEU2+ ]/pUK421 (CEN TRPI+ GAL-H4-2 +) molecule subtype: Nucleosomal DNA from the indicated test strain
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Growth protocol |
For the histone depletion time course, the UKY403 strain harboring a deletion of both Histone H4 genes (HHF1, HHF2), a plasmid with the GAL1 promoter driving expression of Histone H4 (HHF2) and a plasmid with the GAL1 promoter driving expression of Histone H4 (HHF2), was grown in YP with 2% galactose until mid log phase. Cells were then collected by centrifugation and transferred to YPD for 0, 1⁄4,1, 3, 5, and 6 hrs. After each time point, samples were taken for nucleosome and expression analysis.
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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
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Organism |
Saccharomyces cerevisiae |
Characteristics |
strain: BY4741, MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
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Growth protocol |
For the histone depletion time course, the UKY403 strain harboring a deletion of both Histone H4 genes (HHF1, HHF2), a plasmid with the GAL1 promoter driving expression of Histone H4 (HHF2) and a plasmid with the GAL1 promoter driving expression of Histone H4 (HHF2), was grown in YP with 2% galactose until mid log phase. Cells were then collected by centrifugation and transferred to YPD for 0, 1⁄4,1, 3, 5, and 6 hrs. After each time point, samples were taken for nucleosome and expression analysis.
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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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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 after 3 hours of histone H4 depletion 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 |
GSM1093323_H4-depletion_180min.CEL.gz |
33.0 Mb |
(ftp)(http) |
CEL |
GSM1093323_H4-depletion_180min.bar.gz |
18.7 Mb |
(ftp)(http) |
BAR |
Processed data provided as supplementary file |
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