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| Status |
Public on Jan 01, 2012 |
| Title |
rtt101_BrdU_HU_60_min |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
BrdU-IP from rtt101 deletion mutant cells in HU, 60 min
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: W303
antibody: BrdU
vendor/catalog: BDBioscience 555627
genotype/variation: rtt101 deletion mutant cells
time point: 60 min
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| Growth protocol |
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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| Channel 2 |
| Source name |
Genomic DNA from Saccharomyces cerevisiae
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: W303
fraction: input
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| Growth protocol |
G1-synchronised cells were released into S phase in the presence of Hydroxyurea (200mM) and BrdU (400µg/ml) for the indicated time.
|
| 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.
|
| Label |
biotin
|
| 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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| Data processing |
results file descriptions: signal bar files generated for each strains contains log2 (IP/Input) for signal intensity.
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| Submission date |
Nov 14, 2011 |
| Last update date |
Jan 01, 2012 |
| 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 (1) |
| GSE33686 |
dNTP pools determine fork progression and origin usage under replication stress |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM832849_JP012_WCEA.CEL.gz |
32.1 Mb |
(ftp)(http) |
CEL |
| GSM832849_LC029_WCEA.CEL.gz |
34.8 Mb |
(ftp)(http) |
CEL |
| GSM832849_rtt101_LC034_IPB.CEL.gz |
32.2 Mb |
(ftp)(http) |
CEL |
| GSM832849_rtt101_LC034_IPB_signal.bar.gz |
15.5 Mb |
(ftp)(http) |
BAR |
| Processed data provided as supplementary file |
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