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Sample GSM5473837 Query DataSets for GSM5473837
Status Public on Jul 01, 2023
Title HeLa-S3 siGLN3_2
Sample type genomic
 
Channel 1
Source name nascent DNA from early S-phase
Organism Homo sapiens
Characteristics cell line: HeLa-S3
knockdown: siGLN3
molecule subtype: nascent DNA
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 immunoprecipited 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 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 nascent DNA from late S-phase
Organism Homo sapiens
Characteristics cell line: HeLa-S3
knockdown: siGLN3
molecule subtype: nascent DNA
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 immunoprecipited 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 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.
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 Jul 26, 2021
Last update date Jul 01, 2023
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)
GSE180865 GNL3/nucleostemin links DNA replication homeostasis and replication forks stability

Data table header descriptions
ID_REF
VALUE log-ratio representing late vs early replication timing (Cy5/Cy3)

Data table
ID_REF VALUE
1 1.265079468e-003
2 1.000000000e+000
3 1.000000000e+000
4 1.000000000e+000
5 1.000000000e+000
6 1.000000000e+000
7 1.000000000e+000
8 1.000000000e+000
9 1.000000000e+000
10 1.000000000e+000
11 1.000000000e+000
12 1.000000000e+000
13 1.000000000e+000
14 1.000000000e+000
15 1.000000000e+000
16 1.000000000e+000
17 1.000000000e+000
18 1.000000000e+000
19 1.000000000e+000
20 1.000000000e+000

Total number of rows: 180880

Table truncated, full table size 4130 Kbytes.




Supplementary file Size Download File type/resource
GSM5473837_HeLa-S3_siGLN3_2.txt.gz 52.2 Mb (ftp)(http) TXT
Processed data included within Sample table

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