|
| Status |
Public on Mar 31, 2008 |
| Title |
Normal liver tissue 5-meC, promoter array 2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Normal liver tissue
|
| Organism |
Homo sapiens |
| Characteristics |
ChIP sample immunoprecipitated with 5-meC antibody
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed by shaking at 50C o/n in digestion buffer (100mM NaCl; 10mM Tris Cl, pH 8; 25mM EDTA, pH 8; 0.5% SDS; 0.1 mg/ml proteinase K) and genomic DNA was extracted with phenol-chloroform/isoamyl alcohol. ChIP assay with 5-Methylcytidine antibody was performed using ChIP-IT Express kit (Active Motif, cat#53008)
|
| 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 |
Normal liver tissue
|
| Organism |
Homo sapiens |
| Characteristics |
Total input DNA (control)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed by shaking at 50C o/n in digestion buffer (100mM NaCl; 10mM Tris Cl, pH 8; 25mM EDTA, pH 8; 0.5% SDS; 0.1 mg/ml proteinase K) and genomic DNA was extracted with phenol-chloroform/isoamyl alcohol. DNA was sonicated.
|
| Label |
Cy3
|
| Label protocol |
DNA sample (1 μg) was denatured in the presence of 5'-Cy3-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 |
12 ug of the Cy5-labeled ChIP sample and 12 ug of the Cy3-labeled total sample were mixed, dried down, and resuspended in 40 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 |
No additional description
|
| 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 |
Feb 12, 2008 |
| Last update date |
Mar 12, 2008 |
| 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 |
GPL3930 |
| Series (1) |
| GSE10504 |
Using ChIP-chip technology to reveal common principles of transcriptional repression in normal and cancer cells |
|
