|
| Status |
Public on May 19, 2021 |
| Title |
U2OS replication timing in APH condition - Rep2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Nascent DNA from early S phase
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: U2OS
tissue: Osteosarcoma
condition: APH condition
timing: T0 replication timing
phase: early S phase
|
| Treatment protocol |
Aphidicolin (Sigma aldrich) was used at 0.2µM during appropriate duration (see Table S2)
|
| Growth protocol |
Each cells were grown in appropriate culture condition (see material and method)
|
| 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 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 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 |
Nascent DNA from late S phase
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: U2OS
tissue: Osteosarcoma
condition: APH condition
timing: T0 replication timing
phase: late S phase
|
| Treatment protocol |
Aphidicolin (Sigma aldrich) was used at 0.2µM during appropriate duration (see Table S2)
|
| Growth protocol |
Each cells were grown in appropriate culture condition (see material and method)
|
| 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 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 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 (Agilent C-scanner) using a resolution of 3 µm and the autofocus option.
|
| Description |
U2OS_t0_APH_Rep2
|
| 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.
US92003687*txt raw data file: This file contains the raw values of log ratio for each probe (Cy3/Cy5 = E/L) - column P
*log_ratio.txt file: LOESS SMOOTH of log ratio Early/Late values
To determine the replication domains and do the comparative analysis in different conditions, the online platform specific for replication timing data START-R (Hadjadj et al., 2020) was used, with biological duplicates for each condition. The output bed file gave the list of significantly impacted genomic regions (ADVANCED or DELAYED) and the report of number and percentage of genomic regions impacted.
genome build: hg18
Bed file: Genomic coordinate of regions significant replication timing modifications (ADVANCED or DELAYED) between DMSO and Aphidicolin conditions. Note: K562_DMSO_vs_APH_N+1.bed is empty because there are no significant differences between the two conditions.
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| |
|
| Submission date |
Aug 21, 2020 |
| Last update date |
May 19, 2021 |
| Contact name |
Valérie Bergoglio |
| E-mail(s) |
valerie.bergoglio@inserm.fr
|
| Phone |
0582741655
|
| Organization name |
INSERM UMR1037
|
| Lab |
CRCT
|
| Street address |
2 avenue Hubert Curien
|
| City |
Toulouse cedex 1 |
| ZIP/Postal code |
31037 |
| Country |
France |
| |
|
| Platform ID |
GPL10123 |
| Series (1) |
| GSE156618 |
Replication timing data for six human cell lines under aphidicolin treatment (t0) and after release (N+1) |
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