|
| Status |
Public on Mar 28, 2024 |
| Title |
MCF10A DMSO treated Replicate 3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
MCF10A breast epithelial cells
|
| Organism |
Homo sapiens |
| Characteristics |
gender: female
tumor stage: non tumorigenic
sample type: Nascent DNA from Early S-phase
|
| Treatment protocol |
XL413 (synthesised in-house) was used at a concentration of 10 μM.
|
| Growth protocol |
MCF10A cells were cultured using DMEM supplemented with 5% (v/v) horse serum, 25 ng/ml cholera toxin, 10 μg/ml insulin, 20 ng/ml epidermal growth factor (Peprotech), 500 ng/ml hydrocortisone, 50 U/ml penicillin and 50 μg/ml streptomycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were incubated with 50µM BrdU at 37°C for 90 min. Cells were then collected, washed three times in PBS, fixed in 75% final cold EtOH and stored at -20°C. BrdU labelled cells were incubated with 80 μg/mL Propidium Iodide and with 0.4 mg/ml RNaseA for 1 hr at room temperature. 150,000 cells were sorted in early (S1) and late (S2) S-phase fractions using a Fluorescence Activated Cell Sorting system (FACS Aria Fusion, BD). Immunoprecipitation of neo-synthesized DNA was performed using an anti-BrdU antibody (10 μg, purified mouse Anti-BrdU, BD Biosciences, #347580). Control quantitative PCRs (qPCRs) were performed using oligonucleotides specific of early (BMP1 gene) or late (DPPA2 gene) replicating regions.
|
| Label |
Cy3
|
| Label protocol |
Microarray hybridization requires a minimum of 1000 ng of DNA. To obtain sufficient specific immunoprecipited 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 |
MCF10A breast epithelial cells
|
| Organism |
Homo sapiens |
| Characteristics |
gender: female
tumor stage: non tumorigenic
sampel type: Nascent DNA from late S-phase
|
| Treatment protocol |
XL413 (synthesised in-house) was used at a concentration of 10 μM.
|
| Growth protocol |
MCF10A cells were cultured using DMEM supplemented with 5% (v/v) horse serum, 25 ng/ml cholera toxin, 10 μg/ml insulin, 20 ng/ml epidermal growth factor (Peprotech), 500 ng/ml hydrocortisone, 50 U/ml penicillin and 50 μg/ml streptomycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were incubated with 50µM BrdU at 37°C for 90 min. Cells were then collected, washed three times in PBS, fixed in 75% final cold EtOH and stored at -20°C. BrdU labelled cells were incubated with 80 μg/mL Propidium Iodide and with 0.4 mg/ml RNaseA for 1 hr at room temperature. 150,000 cells were sorted in early (S1) and late (S2) S-phase fractions using a Fluorescence Activated Cell Sorting system (FACS Aria Fusion, BD). Immunoprecipitation of neo-synthesized DNA was performed using an anti-BrdU antibody (10 μg, purified mouse Anti-BrdU, BD Biosciences, #347580). Control quantitative PCRs (qPCRs) were performed using oligonucleotides specific of early (BMP1 gene) or late (DPPA2 gene) replicating regions.
|
| Label |
Cy5
|
| Label protocol |
Microarray hybridization requires a minimum of 1000 ng of DNA. To obtain sufficient specific immunoprecipited 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.
|
| Scan protocol |
Microarrays were scanned with an Agilent's High-Resolution C Scanner using a resolution of 3 µm and the autofocus option.
|
| Description |
Biological replicate 3 of 4 MCF10A DMSO treated cells
|
| 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 START-R software.
|
| |
|
| Submission date |
Nov 29, 2023 |
| Last update date |
Mar 28, 2024 |
| Contact name |
Chrystelle Maric |
| E-mail(s) |
chrystelle.maric@ijm.fr
|
| Phone |
0157278074
|
| Organization name |
CNRS
|
| Department |
UMR7592 Institut Jacques-Monod
|
| Lab |
Pathology of DNA replication
|
| Street address |
15 rue Hélène Brion
|
| City |
PARIS |
| ZIP/Postal code |
75205 |
| Country |
France |
| |
|
| Platform ID |
GPL10123 |
| Series (1) |
| GSE248981 |
DBF4, not DRF1, is the crucial regulator of CDC7 kinase at replication forks. |
|
