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Sample GSM154624 Query DataSets for GSM154624
Status Public on Jan 13, 2007
Title ES cells primary clone no 4
Sample type genomic
 
Channel 1
Source name ES cells_primary clone_no 4
Organism Mus musculus
Characteristics Strain : (129/Sv x 129/Sv-CP) F1
Gender : male
Passage: 24
Puromycin resistant
Biomaterial provider ES cells were engineered in the laboratory of Dr Guy Sauvageau, IRIC, Université de Montréal, Canada.
Treatment protocol Infection of target cells. ES cells (passage 18) were infected with the anchor retrovirus A1 produced by the GP+E-86 ecotropic packaging cell line. Viral titers were kept low to ensure low infection rate and to minimize chances of multiple integrations. 24h infections were carried out using 4ug ml-1 of polybren (Sigma). Fresh media was added the next day and puromycin (1.5 ug ml-1) selection started 48h after infection. 7-9 days later, colonies were isolated in 96 wells plates and expanded in presence of puromycin (primary clone). Fractions of these clones were put aside on gelatin coated plates and used to extract DNA.
Growth protocol Prior to DNA extraction, ES cells were grown on gelatin coated dishes, in a media (DMEM high glucose with L-glutamine and pyruvate (Invitrogen), 15% fetal calf serum (Invitrogen),1.5 x 10-4 M alpha-monothioglycerol (Sigma) and 1x10-4 M non-essential amino acids (Invitrogen)) supplemented with 1000 U ml-1 of ESGRO (Chemicon) or leukemia inhibitory factor (LIF, conditioned media from transfected COS cells).
Extracted molecule genomic DNA
Extraction protocol Genomic DNA was isolated using DNAzol according to the manufacturer instructions (Invitrogen).
Label Cy5
Label protocol 1μg of test sample genomic DNA was fluorescently labeled using the BioArray CGH Labeling System (Enzo Life Sciences). Initially, the DNA was denatured in the presence of the random primer at 99ºC for 10 minutes in a thermalcycler, and then quickly cooled to 4ºC. The tubes were transferred to ice and labeling occurred with the addition of dNTP-cyanine 5 mix and Klenow. Incubation took place overnight at 37ºC in a thermalcycler. The unincorporated nucleotides were removed using a QIAquick PCR purification column (Qiagen) and the labeled probe was eluted with 2 x 25 ul washes.
 
Channel 2
Source name normal mouse
Organism Mus musculus
Characteristics Strain: C57BL/6J
Gender: female
Biomaterial provider Microarray and Genomics Facility of the Roswell Park Cancer Institute (Buffalo, NY)
Treatment protocol no treatment
Extracted molecule genomic DNA
Extraction protocol DNA was extracted using the Quiagen FlexiGene DNA kit, according to the manufacturer instructions.
Label Cy3
Label protocol 1μg of reference genomic DNA was fluorescently labeled using the BioArray CGH Labeling System (Enzo Life Sciences). Initially, the DNA was denatured in the presence of the random primer at 99ºC for 10 minutes in a thermalcycler, and then quickly cooled to 4ºC. The tubes were transferred to ice and labeling occurred with the addition of dNTP-cyanine 3 mix and Klenow. Incubation took place overnight at 37ºC in a thermalcycler. The unincorporated nucleotides were removed using a QIAquick PCR purification column (Qiagen) and the labeled probe was eluted with 2 x 25 ul washes.
 
 
Hybridization protocol Prior to hybridization, the test and reference probes were combined with 100 ug mouse fluorimetric Cot-1 (Invitrogen) and ethanol precipitated. Following centrifugation, the probes were resuspended in 110ul SlideHyb Buffer #3 (Ambion) containing 5ul of 100μg/ul Yeast tRNA (Invitrogen), heated to 95ºC for 5 minutes and incubated for 30 minutes at 37ºC to block repetitive elements on the probe. Hybridization was performed for 16 hours at 55ºC using a GeneMachine hybridization station (Genomic Solutions, Inc.) as described (Cowell, J. K. and N. J. Nowak. 2003. High-resolution analysis of genetic events in cancer cells using bacterial artificial chromosome arrays and comparative genome hybridization. Adv Cancer Res 90:91-125). After hybridization, the slides were automatically washed in the GeneMachines station with reducing concentrations of SSC and SDS. The final wash was 30 seconds in 0.1X SSC, followed by a two second 100 % ethanol dip. The slides were spun dry and scanned immediately at 5µm resolution using a GenePix 4200A laser scanner (Axon Inc.)
Scan protocol Slides were scanned on a GenePix 4200A laser Scanner (Axon Inc.) using GenePix Pro V5.0. Both lasers were set to 100% and the PMT was set to 500 for cy3 and 450 for cy5.
Description no additional information
Data processing Images were analyzed using Imagene 6.0 (BioDiscovery Inc.). The output of Image analysis was further processed by in-house developed programs. The log2 ratios of the test/control (values are not background subtracted) were normalized using a subgrid loess, with all flagged spots and clones on the sex chromosome are given a weight of zero. The mean of the replicate measures was taken and the final normalized log2 ratio was plotted against its genomic location.
 
Submission date Jan 10, 2007
Last update date Jan 12, 2007
Contact name Guy Sauvageau
E-mail(s) guy.sauvageau@gmail.com
Organization name IRIC
Department Université de Montréal
Lab Laboratory of Molecular Genetics of Stem Cells
Street address Marcelle-Coutu Building 2900 Boul. Edouard Montpetit Quai 20
City Montreal
State/province Quebec
ZIP/Postal code H3T 1J4
Country Canada
 
Platform ID GPL4714
Series (1)
GSE6706 A retroviral strategy that efficiently creates chromosomal deletions in mammalian cells

Data table header descriptions
ID_REF
COUNT The number of replicates used to calculate the data
VALUE Log2 ratio: the log2 of the test/control
Ratio The linear ratio of the test/control
A Measure of brightness: (log2 cy3 + log2 cy5)/2

Data table
ID_REF COUNT VALUE Ratio A
RP23-271O17 3 0.0933 1.0668 8.6532
RP23-232J11 0
RP23-320L20 0
RP23-445L18 3 0.24 1.181 10.0587
RP23-131C23 0
RP23-32F6 3 0.0167 1.0116 8.6835
RP23-297K4 3 0.0933 1.0668 8.9339
RP23-422K10 0
RP23-69J22 0
RP23-294B12 3 0.4067 1.3256 10.523
RP23-410H10 0
RP23-315F19 3 0.09 1.0644 9.8809
RP23-144B15 0
RP23-480F21 3 0.28 1.2142 9.4186
RP23-280K9 3 0.1233 1.0892 9.5146
RP23-291G6 0
RP23-272K15 0
RP23-303L3 0
RP23-50B16 3 0.2133 1.1594 9.3129
RP23-32B12 0

Total number of rows: 6507

Table truncated, full table size 145 Kbytes.




Supplementary file Size Download File type/resource
GSM154624_Cy3.tif.gz 15.1 Mb (ftp)(http) TIFF
GSM154624_Cy3.txt.gz 856.1 Kb (ftp)(http) TXT
GSM154624_Cy5.tif.gz 15.5 Mb (ftp)(http) TIFF
GSM154624_Cy5.txt.gz 857.5 Kb (ftp)(http) TXT

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