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Sample GSM4195543 Query DataSets for GSM4195543
Status Public on Nov 28, 2019
Title Manual2 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

Data table header descriptions
ID_REF
VALUE Log2-ratio representing early versus late replication timing

Data table
ID_REF VALUE
1 -2.55E-01
2 0.00E+00
3 0.00E+00
4 0.00E+00
5 0.00E+00
6 0.00E+00
7 0.00E+00
8 0.00E+00
9 0.00E+00
10 0.00E+00
11 0.00E+00
12 0.00E+00
13 0.00E+00
14 0.00E+00
15 0.00E+00
16 0.00E+00
17 0.00E+00
18 -1.34E-01
19 0.00E+00
20 0.00E+00

Total number of rows: 180880

Table truncated, full table size 2809 Kbytes.




Supplementary file Size Download File type/resource
GSM4195543_Manual2_U2OS_replication_timing.txt.gz 52.8 Mb (ftp)(http) TXT
Processed data included within Sample table

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