NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM4734956 Query DataSets for GSM4734956
Status Public on May 19, 2021
Title RPE-1 replication timing in DMSO condition - Rep2
Sample type genomic
 
Channel 1
Source name Nascent DNA from early S phase
Organism Homo sapiens
Characteristics cell line: RPE-1
tissue: Retina
condition: DMSO 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: RPE1
tissue: Retina
condition: DMSO 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 RPE_t0_DMSO_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.
 
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)

Supplementary file Size Download File type/resource
GSM4734956_US92003687_252206079791_S01_CGH_1105_RPE_DMSO_Rep2.txt.gz 51.9 Mb (ftp)(http) TXT
Processed data are available on Series record

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap