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Sample GSM1015445 Query DataSets for GSM1015445
Status Public on Jul 01, 2014
Title IMR90 Apoptosis (GPL9777)
Sample type genomic
 
Channel 1
Source name fetal lung fibroblast
Organism Homo sapiens
Characteristics gender: female
cell line: IMR90
sample type: DNA fragment ~ 4 kb
Treatment protocol Apoptosis was induced by staurosporine (Cell Signaling Technology, Inc., Danvers, USA), which was dissolved in dimethyl sulfoxide (DMSO, Sigma, Saint Louis, USA) to a stock concentration of 1 mmol/L. About 2 x 10^6 cells were exposed to either 1 µmol/L staurosporine/0.1%DMSO or 0.1% DMSO alone as a control for 4 hours at 37°C.
Growth protocol Both cell lines were cultured in Eagle´s minimum essential medium (EMEM) supplemented with 10% fetal bovine serum, 2 mM UltraGlutamine 1, 1 mM sodium pyruvate and 100units/ml penicillin/streptomycin. The fibroblasts were maintained at 37°C with a humidified atmosphere of 5% CO2 and ambient oxygen.
Extracted molecule genomic DNA
Extraction protocol ChIP was done according to the Transcription Factor ChIP kit protocol (Diagenode, Liège, Belgium). In brief, lysed cells were sonicated for 60 minutes using the Bioruptor UCD-200 device (Diagenode, Liège, Belgium), followed by overnight incubation of 1x106 cells with 5 µg of antibodies against unmodified histone H3 (ab1791; Abcam, Cambridge, UK) and histone H4 lysine 8 acetylation (pAb-103-050; Diagenode, Liège, Belgium), respectively. The subsequent chromatin reverse crosslinking, elution and purification of ChIP and input DNA were done employing the IPure Kit (Diagenode, Liège, Belgium). Analysis of apoptotic DNA: after treatment cells were washed once in PBS, detached by five minutes treatment with trypsin-EDTA and the cell pellets were stored at -80°. For DNA isolation cells were treated with lysis buffer (0.40 M Tris-HCl pH 8.0, 0.06 M Na-EDTA, 0.15 M NaCl, 1% SDS) and RNA was digested for 1 hour at 37°C using 15 µg/mL RNase A. 1 M sodium perchlorate and one volume chloroform were added to deproteinize cell lysates. After phase separation and collection of the aqueous phase DNA was precipitated. 3 µg of genomic DNA were loaded onto a 1% low melt agarose gel, electrophoresed for 90 minutes at 120 V and stained with ethidium bromide for 30 minutes. DNA bands responding to non-fragmented (> 48 kb) and fragmented DNA (approximately 4 kb) were cut with a scalpel and collected. For the extraction of DNA from the gel pieces β-Agarase I (New England Biolabs, Ipswich, USA) was used according to the manufacturer´s protocol.
Label Cy3
Label protocol Agilent Genomic DNA Enzymatic Labeling Kit
 
Channel 2
Source name fetal lung fibroblast
Organism Homo sapiens
Characteristics gender: female
cell line: IMR90
sample type: DNA fragment > 48 kb
Treatment protocol Apoptosis was induced by staurosporine (Cell Signaling Technology, Inc., Danvers, USA), which was dissolved in dimethyl sulfoxide (DMSO, Sigma, Saint Louis, USA) to a stock concentration of 1 mmol/L. About 2 x 10^6 cells were exposed to either 1 µmol/L staurosporine/0.1%DMSO or 0.1% DMSO alone as a control for 4 hours at 37°C.
Growth protocol Both cell lines were cultured in Eagle´s minimum essential medium (EMEM) supplemented with 10% fetal bovine serum, 2 mM UltraGlutamine 1, 1 mM sodium pyruvate and 100units/ml penicillin/streptomycin. The fibroblasts were maintained at 37°C with a humidified atmosphere of 5% CO2 and ambient oxygen.
Extracted molecule genomic DNA
Extraction protocol ChIP was done according to the Transcription Factor ChIP kit protocol (Diagenode, Liège, Belgium). In brief, lysed cells were sonicated for 60 minutes using the Bioruptor UCD-200 device (Diagenode, Liège, Belgium), followed by overnight incubation of 1x106 cells with 5 µg of antibodies against unmodified histone H3 (ab1791; Abcam, Cambridge, UK) and histone H4 lysine 8 acetylation (pAb-103-050; Diagenode, Liège, Belgium), respectively. The subsequent chromatin reverse crosslinking, elution and purification of ChIP and input DNA were done employing the IPure Kit (Diagenode, Liège, Belgium). Analysis of apoptotic DNA: after treatment cells were washed once in PBS, detached by five minutes treatment with trypsin-EDTA and the cell pellets were stored at -80°. For DNA isolation cells were treated with lysis buffer (0.40 M Tris-HCl pH 8.0, 0.06 M Na-EDTA, 0.15 M NaCl, 1% SDS) and RNA was digested for 1 hour at 37°C using 15 µg/mL RNase A. 1 M sodium perchlorate and one volume chloroform were added to deproteinize cell lysates. After phase separation and collection of the aqueous phase DNA was precipitated. 3 µg of genomic DNA were loaded onto a 1% low melt agarose gel, electrophoresed for 90 minutes at 120 V and stained with ethidium bromide for 30 minutes. DNA bands responding to non-fragmented (> 48 kb) and fragmented DNA (approximately 4 kb) were cut with a scalpel and collected. For the extraction of DNA from the gel pieces β-Agarase I (New England Biolabs, Ipswich, USA) was used according to the manufacturer´s protocol.
Label Cy5
Label protocol Agilent Genomic DNA Enzymatic Labeling Kit
 
 
Hybridization protocol see manufacturer website: http://www.chem.agilent.com/Library/usermanuals/Public/G4410-90010_CGH_Enzymatic_Protocol_v6.2.pdf
Scan protocol scanned on an Agilent DNA Microarray Scanner G2565BA, Scan resolution: 2µm, Scan region: 61 x 21.6 mm, Green PMT: 100%, Red PMT: 100%
Description Regional preferences in apoptotic DNA degradation were determined by co-hybridizing high molecular (> 48 kb) and degraded apoptotic DNA (~4 kb), which we have extracted from 1% low melting agarose and amplified by means of the GenomePlex Whole Genome Amplification Kit .
Data processing TIFF image processing with Agilent Feature Extraction v10.5.1.1, CGH_105_Dec08 protocol. Data visualization and further analysis was performed with GenomeCATPro 1.3.1 (Tebel et al., manuscript in preparation; http://www.molgen.mpg.de/~abt_rop/molecular_cytogenetics/CGHPRO.html) and the UCSC genome browser (https://genome.ucsc.edu/index.html).
 
Submission date Oct 04, 2012
Last update date Jul 02, 2014
Contact name Reinhard Ullmann
E-mail(s) ullmann@molgen.mpg.de
Phone 00493084131251
Organization name MPIMG
Department Human Molecular Genetics
Lab Molecular Cytogenetics
Street address Ihnestr.73
City Berlin
ZIP/Postal code 14195
Country Germany
 
Platform ID GPL9777
Series (1)
GSE41356 Distribution of segmental duplications in the context of higher order chromatin organisation of human chromosome 7

Data table header descriptions
ID_REF
VALUE -INV_VALUE: normalized log10 ratio Cy3/Cy5
INV_VALUE normalized log10 ratio Cy5/Cy3

Data table
ID_REF VALUE INV_VALUE
1 -0.00409627 4.096269804e-003
2 0.000000000e+000 0.000000000e+000
3 0.321715 -3.217147255e-001
4 0.0453044 -4.530436096e-002
5 -0.0633902 6.339021991e-002
6 -0.024468 2.446800331e-002
7 -0.076352 7.635200305e-002
8 -0.0914464 9.144637847e-002
9 0.11729 -1.172897253e-001
10 0.433957 -4.339573224e-001
11 0.0361104 -3.611043238e-002
12 -0.278534 2.785336241e-001
13 -0.107791 1.077912724e-001
14 0.0259844 -2.598441237e-002
15 0.0541311 -5.413114573e-002
16 -0.0621257 6.212573786e-002
17 -0.160753 1.607531073e-001
18 -0.401928 4.019278974e-001
19 -0.395325 3.953253954e-001
20 -0.383398 3.833982464e-001

Total number of rows: 420288

Table truncated, full table size 13989 Kbytes.




Supplementary file Size Download File type/resource
GSM1015445_IMR90_Apoptosis.txt.gz 120.4 Mb (ftp)(http) TXT
Processed data included within Sample table
Processed data provided as supplementary file

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