|
| Status |
Public on Apr 09, 2010 |
| Title |
HEK293 5-azaC H3K9me3 [ChIP-chip] |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
H3K9me3 ChIP DNA from HEK293 cells treated with 5 uM 5-azacytidine for 8 days
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: 5uM 5-azacytidine for 8 days
antibody: H3K9me3
|
| 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
|
| 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.
|
| |
|
| 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.
|
| |
|
| |
| 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.
|
| Scan protocol |
The arrays were scanned at 5-μm resolution using the GenePix 4000B scanner (Axon Instruments)
|
| Description |
Antibody: rabbit polyclonal H3K9me3 IgG (Abcam cat # ab8898; 3 ug per ChIP assay). No secondary
|
| 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
|
| |
|
| Submission date |
Mar 02, 2010 |
| Last update date |
Apr 09, 2010 |
| Contact name |
Vitalina Komashko |
| E-mail(s) |
vitalina.komashko@sagebase.org
|
| Organization name |
Sage Bionetworks
|
| Street address |
1100 Fairview Ave N
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL6603 |
| Series (1) |
| GSE20598 |
5-azacytidine treatment reorganizes genomic histone modification patterns |
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