|
| Status |
Public on Apr 09, 2010 |
| Title |
HEK293 aa 5-meC [ChIP-chip] |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
5-meC MeDIP DNA from HEK293 cells treated with 50% acetic acid (control)
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: 50% acetic acid
|
| Treatment protocol |
Cells were treated with 50% acetic acid for the same time periods as the cells treated with 5-azacytidine. Amount of acetic acid was the same by volume as used for 5-azacytidine
|
| 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 50% acetic acid
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: 50% acetic acid
|
| Treatment protocol |
Total input DNA from HEK293 cells treated with 50% acetic acid
|
| Growth protocol |
Cells were treated with 50% acetic acid for the same time periods as the cells treated with 5-azacytidine. Amount of acetic acid was the same by volume as used for 5-azacytidine
|
| 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 |
Extraction protocol specific to MeDIP samples: genomic DNA was extracted by shaking cells in digestion buffer (100mM NaCl, 10mM TrisCl, pH 8, 25mM EDTA, pH 8, 0.5% SDS) and 0.1 mg/ml Proteinase K for 12-18 hours at 50C and purified using phenol-chlorophorm extraction method. Extracted DNA was sonicated to an average size of 500 bp, denatured at 95C for 10 min and quickly chilled on ice. Immunoprecipitation of methylated DNA was performed with 4 ug mouse monoclonal 5-Methylcytidine (Eurogentec cat# BI-MECY-0100) antibody per 4 ug of sonicated genomic DNA using the same buffers as for the ChIP assays. The secondary rabbit anti-mouse IgG was purchased from MP Biomedicals (cat # 55436). Incubation with primary antibody was performed for 2 hours and for 1 hour with the secondary antibody. Antibody-DNA complexes were captured using Staph A cells, then the DNA was washed, eluted and incubated with Proteinase K. The non-specific rabbit IgG used as a negative control in the ChIP and MeDIP assays was purchased from Alpha Diagnostics (cat # 20009-5).
|
| 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 |
|
