|
| Status |
Public on Nov 28, 2019 |
| Title |
Automated1 U2OS replication timing |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
nascent DNA from early S-phase
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: U2OS
phase: Early S-phase
|
| Growth protocol |
Each cells grown in culture condition defined by ATCC.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were incubated with BrdU (50µM) for two hours, 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 immunoprecipitated by BrdU antibodies (Anti BrdU Pure, BD Biosciences, # 347580). To control the quality of enrichment of early and late fractions in S1 and S2, qPCR was performed with BMP1 oligonucleotides (early control) and with Dppa2 oligonucleotides (late control).
|
| Label |
Cy3
|
| Label protocol |
Microarray hybridization requires a minimum of 1000 ng of DNA and to obtain sufficient specific immunoprecipitated DNA, whole genome amplification (WGA) was conducted (Seq-plex, 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 |
nascent DNA from late S-phase
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: U2OS
phase: Late S-phase
|
| Growth protocol |
Each cells grown in culture condition defined by ATCC.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were incubated with BrdU (50µM) for two hours, 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 immunoprecipitated by BrdU antibodies (Anti BrdU Pure, BD Biosciences, # 347580). To control the quality of enrichment of early and late fractions in S1 and S2, qPCR was performed with BMP1 oligonucleotides (early control) and with Dppa2 oligonucleotides (late control).
|
| Label |
Cy5
|
| Label protocol |
Microarray hybridization requires a minimum of 1000 ng of DNA and to obtain sufficient specific immunoprecipitated DNA, whole genome amplification (WGA) was conducted (Seq-plex, 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 or 8 X44K human microarray.
|
| Scan protocol |
Microarrays were scanned with an Agilent's High-Resolution C Scanner using a resolution of 3 µm and the autofocus option.
|
| Description |
Nascent DNA
|
| 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 |
Nov 27, 2019 |
| Last update date |
Nov 28, 2019 |
| 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) |
| GSE141122 |
New, easy, quick and efficient DNA replication timing analysis by high-throughput approaches |
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