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Sample GSM1093323 Query DataSets for GSM1093323
Status Public on May 22, 2013
Title H4-depletion_180min [nucleosome profile]
Sample type genomic
 
Channel 1
Source name UKY403 after 3 hours of histone H4 depletion
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
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.
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.
 
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 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.
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.
 
 
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.
Scan protocol Arrays were scanned in an Affymetrix 7G scanner.
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
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.
 
Submission date Mar 05, 2013
Last update date May 22, 2013
Contact name Harm van Bakel
E-mail(s) [email protected]
Organization name Mount Sinai School of Medicine
Department Genetics and Genomic Sciences
Lab Bakel Lab
Street address One Gustave L. Levy Place, Box 1498
City New York
State/province New York
ZIP/Postal code 10029
Country USA
 
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

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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