|
Status |
Public on May 22, 2013 |
Title |
RPB2_tet [nucleosome profile] |
Sample type |
genomic |
|
|
Channel 1 |
Source name |
RPB2 Tet promotor-shutoff strain
|
Organism |
Saccharomyces cerevisiae |
Characteristics |
strain: BY4741, MATa his3Δ1 leu2Δ0 met15Δ0 URA3::CMV-tTA genotype/variation: RPB2 Tet promotor-shutoff strain molecule subtype: Nucleosomal DNA from the indicated test strain
|
Growth protocol |
For Tet promoter-shutoff strains, doxycyline was added at 10μg/ml for 24 h in order to obtain down-regulation for the gene of interest. Cells were then diluted to an OD600 of 0.2 and grown in the presence of doxycycline (10μg/ml) until mid-log phase.
|
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 Tet promoter-shutoff strains, doxycyline was added at 10μg/ml for 24 h in order to obtain down-regulation for the gene of interest. Cells were then diluted to an OD600 of 0.2 and grown in the presence of doxycycline (10μg/ml) until mid-log phase.
|
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 for RPB2 Tet promotor-shutoff strain 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.
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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]
|
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 |
|