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Sample GSM5401040 Query DataSets for GSM5401040
Status Public on Aug 20, 2021
Title MEF-Rev3+/+_p60
Sample type genomic
 
Channel 1
Source name nascent DNA from early S-phase
Organism Mus musculus
Characteristics cell line: MEF
passage: 60
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 Mus musculus
Characteristics cell line: MEF
passage: 60
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 4x 180K mouse 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
MEF-Rev3+/+_p6_replication timing
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 (Hadjadj D, Denecker T, Guérin E, Kim SJ, Fauchereau F, Baldacci G, Maric C, Cadoret JC. Efficient, quick and easy-to-use DNA replication timing analysis with START-R suite. NAR Genom Bioinform. 2020 Jun 19;2(2):lqaa045. doi: 10.1093/nargab/lqaa045. PMID: 33575597; PMCID: PMC7671386.).
 
Submission date Jun 25, 2021
Last update date Aug 20, 2021
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 GPL10449
Series (1)
GSE178927 DNA polymerase zeta contributes to heterochromatin replication to prevent genome instability

Data table header descriptions
ID_REF
VALUE log-ratio representing early versus late replication timing (i.e., LogRatio column from Agilent Feature Extraction file)

Data table
ID_REF VALUE
1 3.706003788e-001
2 -4.340894358e-001
3 -4.958164430e-001
4 -6.127126310e-001
5 -7.131613311e-001
6 -7.207135939e-001
7 -7.491038650e-001
8 -6.701578153e-001
9 -8.581653237e-001
10 -8.224981370e-001
11 -7.287417889e-001
12 -7.815137339e-001
13 -6.235222930e-001
14 -4.414836423e-001
15 -6.093499819e-001
16 -4.828085395e-001
17 -4.353025531e-001
18 -1.442526330e-002
19 -4.332119516e-002
20 0.000000000e+000

Total number of rows: 180880

Table truncated, full table size 4225 Kbytes.




Supplementary file Size Download File type/resource
GSM5401040_Mef-Rev3++_p60.txt.gz 52.3 Mb (ftp)(http) TXT
Processed data included within Sample table

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