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| Status |
Public on Mar 26, 2014 |
| Title |
hos2 |
| Sample type |
genomic |
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| Source name |
hos2
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| Organism |
Saccharomyces cerevisiae W303 |
| Characteristics |
antibody: anti-BrdU IgG1 (BD Biosciences)
treatment(s): HU
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| Treatment protocol |
Samples were collected in early S phase +/- 200 mM HU (hydroxyurea).
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| Growth protocol |
Cells were grown on YPD at 25°C and were arrested in G1 with alpha-factor. G1-synchronised cells were released into S phase in the presence of Hydroxyurea (200mM) and BrdU (400µg/ml) for the indicated time.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For immunoprecipitation of BrdU-labelled DNA, 1.5x109 cells were lysed for 5 x 2 minutes in NIB buffer (17 % glycerol, 50 mM MOPS buffer, 150mM potassium acetate, 2 mM magnesium chloride, 500 µM spermidine and 150 µM spermine, pH7.2) with Zirconium beads on a Vibrax (VXR basic, Ika) at 4C. DNA was isolated using Qiagen Genomic DNA extraction kit and sonicated to reach an average DNA size of 300-600 bp. For each immunoprecipitation, 10 µl of mouse anti-BrdU IgG1 (BDBioscience 555627) was prebound to Dynabeads M-280 Sheep anti-mouse IgG (Invitrogen 112.02) and added to the denatured purified DNA. For immunoprecipitation of BrdU-labelled DNA, 1.5x109 cells were lysed for 5 x 2 minutes in NIB buffer (17 % glycerol, 50 mM MOPS buffer, 150mM potassium acetate, 2 mM magnesium chloride, 500 µM spermidine and 150 µM spermine, pH7.2) with Zirconium beads on a Vibrax (VXR basic, Ika) at 4C. DNA was isolated using Qiagen Genomic DNA extraction kit and sonicated to reach an average DNA size of 300-600 bp. For each immunoprecipitation, 10 µl of mouse anti-BrdU IgG1 (BDBioscience 555627) was prebound to Dynabeads M-280 Sheep anti-mouse IgG (Invitrogen 112.02) and added to the denatured purified DNA. For immunoprecipitation of BrdU-labelled DNA, 1.5x109 cells were lysed for 5 x 2 minutes in NIB buffer (17 % glycerol, 50 mM MOPS buffer, 150mM potassium acetate, 2 mM magnesium chloride, 500 µM spermidine and 150 µM spermine, pH7.2) with Zirconium beads on a Vibrax (VXR basic, Ika) at 4C. DNA was isolated using Qiagen Genomic DNA extraction kit and sonicated to reach an average DNA size of 300-600 bp. For each immunoprecipitation, 10 µl of mouse anti-BrdU IgG1 (BDBioscience 555627) was prebound to Dynabeads M-280 Sheep anti-mouse IgG (Invitrogen 112.02) and added to the denatured purified DNA. For immunoprecipitation of BrdU-labelled DNA, 1.5.109 cells were lysed for 5 x 2 min in NIB buffer (17% glycerol, 50 mM MOPS buffer, 150 mM potassium acetate, 2 mM magnesium chloride, 500 µM spermidine and 150 µM spermine, pH 7.2) with zirconium beads on a vibrax (VXR basic, IKA) at 4°C. DNA was isolated using Qiagen Genomic DNA extraction kit and sonicated to reach an average DNA size of 300-600 bp. For each immunoprecipitation, 10µl of mouse anti-BrdU IgG1 (BD Biosciences) was prebound to Dynabeads M-280 Sheep anti-mouse IgG (Invitrogen) and added to the denaturated purified DNA.
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| Label |
Biotin
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| Label protocol |
7 µg of amplified DNA was fragmented and labelled using the GeneChip WT Double-Stranded DNA Terminal Labelling kit (Affymetrix, PN 900812) following manufacturer's recommendations. 7 µg of amplified DNA was fragmented and labelled using the GeneChip WT Double-Stranded DNA Terminal Labelling kit (Affymetrix, PN 900812) following manufacturer recommendations.
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| Hybridization protocol |
DNA was hybridized to Affymetrix Genechip S. cerevisiae Tiling 1.0R Array (5 bpresolution, Affymetrix PN 900645) using the GeneChip Hybridization, Wash, and Stain Kit (Affymetrix, PN 900720).
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| Scan protocol |
Tiling arrays were scanned with the GeneChip scanner 3000 7G
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| Description |
hos2_signal.bar
HDAC mutants + HU
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| Data processing |
Signals were normalized with TAS 1.1.02 (Affymetrix) using quantile normalisation. Analysis results were stored in log2 scale for signal intensity and in -10Log10 for p-values. Probe signals were analysed using perfect matches only with a bandwidth of 300. P-value intervals were generated using the following parameters: cutoff: 10-5, max gap: 80 and min run: 40.
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| Submission date |
Mar 25, 2014 |
| Last update date |
Mar 26, 2014 |
| Contact name |
Philippe Pasero |
| E-mail(s) |
ppasero@igh.cnrs.fr
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| Organization name |
Institute of Human Genetics - CNRS
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| Lab |
Pasero
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| Street address |
141 rue de la Cardonille
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| City |
Montpellier |
| ZIP/Postal code |
34396 |
| Country |
France |
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| Platform ID |
GPL7250 |
| Series (2) |
| GSE56171 |
HDACs act on ribosomal DNA to control the yeast replication program and the competition between origins for limiting initiation factors [BrdU-IP-chip] |
| GSE57619 |
HDACs act on ribosomal DNA to control the yeast replication program and the competition between origins for limiting initiation factors |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM1357064_KY002_IPE_hos2.CEL.gz |
27.3 Mb |
(ftp)(http) |
CEL |
| Processed data are available on Series record |
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