|
| Status |
Public on Oct 28, 2014 |
| Title |
control replication timing 2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
neosynthetized DNA in early S phase
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MRC5
genotype/variation: control
growth phase: early S phase
|
| Treatment protocol |
overexpressed PolQ cells are stable cells that overexpress PolQ gene
|
| Growth protocol |
Modified Eagle Medium with GlutaMAX™ I, High glucose, Sodium Pyruvate (Gibco, Life technologies, Paisley, UK), supplemented with 10% Foetal Bovine Serum (Lonza, Basel, CH), penicillin (100U/ml) and streptomycin (100µg/ml) (Gibco) at 37°C, 5% CO2 and 5% O2 (standard culture conditions).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
cells were incubated with BrdU (50µM) for one hour, then collected, washed three times in PBS, fixed in 75% ethanol, and stored at −20°C. Fixed cells were re-suspended in PBS first with RNAse (0.5 mg/ml) and then with propidium iodide (PI) (50 µg/ml) following by an incubation of 30 min at room temperature. 80,000 cells were sorted in two fractions S1 and S2 using INFLUX 500 (Cytopeia purchased by BD Biosciences) corresponding to Early and Late fractions respectively. Neo-synthesized DNA was immunoprecipited by BrdU antibodies (Anti BrdU Pure, BD Biosciences, # 347580) as described by Hiratani et al 2008. To control the quality of enrichment of early and late fractions in S1 and S2, qPCR was performed with CAV2 oligonucleotides (early control) and with bgGRM8 oligonucleotides (late control).
|
| Label |
cy3
|
| Label protocol |
Microarray hybridization requires a minimum of 500 ng of DNA and to obtain sufficient specific immunoprecipited DNA, whole genome amplification was conducted (WGA, Sigma). To be sure that this step does not introduce bias, an after WGA qPCR was performed to confirm the specific enrichment in each fractions S1 and S2. After amplification, early and late neo-synthesized DNA were labeled with Cy3 and Cy5 ULS molecules (Genomic DNA labeling Kit, Agilent) as recommended by the manufacturer.
|
| |
|
| Channel 2 |
| Source name |
neosynthetized DNA in late S phase
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MRC5
genotype/variation: control
growth phase: late S phase
|
| Treatment protocol |
overexpressed PolQ cells are stable cells that overexpress PolQ gene
|
| Growth protocol |
Modified Eagle Medium with GlutaMAX™ I, High glucose, Sodium Pyruvate (Gibco, Life technologies, Paisley, UK), supplemented with 10% Foetal Bovine Serum (Lonza, Basel, CH), penicillin (100U/ml) and streptomycin (100µg/ml) (Gibco) at 37°C, 5% CO2 and 5% O2 (standard culture conditions).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
cells were incubated with BrdU (50µM) for one hour, then collected, washed three times in PBS, fixed in 75% ethanol, and stored at −20°C. Fixed cells were re-suspended in PBS first with RNAse (0.5 mg/ml) and then with propidium iodide (PI) (50 µg/ml) following by an incubation of 30 min at room temperature. 80,000 cells were sorted in two fractions S1 and S2 using INFLUX 500 (Cytopeia purchased by BD Biosciences) corresponding to Early and Late fractions respectively. Neo-synthesized DNA was immunoprecipited by BrdU antibodies (Anti BrdU Pure, BD Biosciences, # 347580) as described by Hiratani et al 2008. To control the quality of enrichment of early and late fractions in S1 and S2, qPCR was performed with CAV2 oligonucleotides (early control) and with bgGRM8 oligonucleotides (late control).
|
| Label |
cy5
|
| Label protocol |
Microarray hybridization requires a minimum of 500 ng of DNA and to obtain sufficient specific immunoprecipited DNA, whole genome amplification was conducted (WGA, Sigma). To be sure that this step does not introduce bias, an after WGA qPCR was performed to confirm the specific enrichment in each fractions S1 and S2. After amplification, early and late neo-synthesized DNA were labeled with Cy3 and Cy5 ULS molecules (Genomic DNA labeling Kit, Agilent) as recommended by the manufacturer.
|
| |
|
| |
| Hybridization protocol |
The hybridization was performed according to the manufacturer instructions on 4× 180K human microarray (SurePrint G3 Human CGH Microarray Kit, 4x180K, AGILENT Technologies, genome reference Hg18) that covers the whole genome with one probe every 13 Kb (11 KB in RefSeq sequences).
|
| Scan protocol |
Microarrays were scanned with an Agilent's High-Resolution C Scanner using a resolution of 2 µm and the autofocus option.
|
| Data processing |
Feature extraction was performed with the Feature Extraction 9.1 software (Agilent technologies). For each experiment, the raw data sets were automatically normalized by the Feature extraction software. Analysis was performed with the Agilent Genomic Workbench 5.0 software.
|
| |
|
| Submission date |
Dec 06, 2013 |
| Last update date |
Oct 29, 2014 |
| Contact name |
Jean-Charles Cadoret |
| E-mail(s) |
jean-charles.cadoret@ijm.fr
|
| Organization name |
CNRS/ Université de Paris
|
| Street address |
15 rue hélène Brion
|
| City |
Paris |
| ZIP/Postal code |
75013 |
| Country |
France |
| |
|
| Platform ID |
GPL10123 |
| Series (1) |
| GSE53070 |
Replication timing of control and overexpressed PolQ fibroblast cells |
|
