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Status |
Public on Apr 09, 2010 |
Title |
HEK293 5-azaC H3K27me3 [ChIP-chip] |
Sample type |
genomic |
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Channel 1 |
Source name |
H3K27me3 ChIP DNA from HEK293 cells treated with 5 uM 5-azacytidine for 8 days
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Organism |
Homo sapiens |
Characteristics |
treatment: 5uM 5-azacytidine for 8 days antibody: H3K27me3
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Treatment protocol |
5-azacytidine was purchased from Sigma (cat # 2385) and dissolved in 50% acetic acid. HEK293 cells were treated with 5uM 5-azacytidine dissolved in medium for 8 days; medium was changed daily.
|
Growth protocol |
DMEM, 10% FBS, 2mM L-glutamine, 1% penicillin/streptomycin
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Extracted molecule |
genomic DNA |
Extraction protocol |
ChIP assay was performed following the ChIP protocol provided athttp://www.genomecenter.ucdavis.edu/farnham and http://genomecenter.ucdavis.edu/expression_analysis.
|
Label |
Cy5
|
Label protocol |
DNA sample (1 μg) was denatured in the presence of 5'-Cy5-labeled random nonamers (TriLink Biotechnologies) and incubated with 100 units (exo-) Klenow fragment (NEB) and dNTP mix [6 mM each in TE buffer (10 mM Tris/1mM EDTA, pH 7.4; Invitrogen)] for 2 h at 37°C. Reaction was terminated by addition of 0.5 M EDTA (pH 8.0), precipitated with isopropanol, and resuspended in water.
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Channel 2 |
Source name |
Total input DNA from HEK293 cells treated with 5uM 5-azacytidine for 8 days
|
Organism |
Homo sapiens |
Characteristics |
treatment: 5uM 5-azacytidine for 8 days antibody: none
|
Treatment protocol |
Total input DNA from HEK293 cells treated with 5uM 5-azacytidine for 8 days
|
Growth protocol |
5-azacytidine was purchased from Sigma (cat # 2385) and dissolved in 50% acetic acid. HEK293 cells were treated with 5uM 5-azacytidine dissolved in medium for 8 days; medium was changed daily.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
ChIP assay was performed following the ChIP protocol provided athttp://www.genomecenter.ucdavis.edu/farnham and http://genomecenter.ucdavis.edu/expression_analysis.
|
Label |
Cy3
|
Label protocol |
DNA sample (1 μg) was denatured in the presence of 5'-Cy5-labeled random nonamers (TriLink Biotechnologies) and incubated with 100 units (exo-) Klenow fragment (NEB) and dNTP mix [6 mM each in TE buffer (10 mM Tris/1mM EDTA, pH 7.4; Invitrogen)] for 2 h at 37°C. Reaction was terminated by addition of 0.5 M EDTA (pH 8.0), precipitated with isopropanol, and resuspended in water.
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Hybridization protocol |
6 ug of the Cy5-labeled ChIP sample and 6 ug of the Cy3-labeled total sample were mixed, dried down, and resuspended in 18 μl of NimbleGen Hybridization Buffer (NimbleGen Systems).After denaturation, hybridization was carried out in a MAUI Hybridization System (BioMicro Systems) for 18 h at 42°C. The arrays were washed using NimbleGen Wash Buffer System (NimbleGen Systems) and dried by centrifugation.
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Scan protocol |
The arrays were scanned at 5-μm resolution using the GenePix 4000B scanner (Axon Instruments)
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Description |
Antibody: rabbit polyclonal H3K27me3 IgG (Millipore cat # 07-449; 3 ug per ChIP assay). No secondary
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Data processing |
Fluorescence intensity raw data was obtained from scanned images of the oligonucleotide tiling arrays using NimbleScan 2.2 extraction software (NimbleGen Systems). For each spot on the array, log2-ratios of the Cy5-labeled test sample versus the Cy3-labeled reference sample were calculated. Then, the biweight mean of this log2 ratio was subtracted from each point; this procedure similar to mean-normalization of each channel
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Submission date |
Mar 02, 2010 |
Last update date |
Apr 09, 2010 |
Contact name |
Vitalina Komashko |
E-mail(s) |
vitalina.komashko@sagebase.org
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Organization name |
Sage Bionetworks
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Street address |
1100 Fairview Ave N
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City |
Seattle |
State/province |
WA |
ZIP/Postal code |
98109 |
Country |
USA |
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Platform ID |
GPL6603 |
Series (1) |
GSE20598 |
5-azacytidine treatment reorganizes genomic histone modification patterns |
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