|
Status |
Public on Mar 31, 2008 |
Title |
Ntera2 RNAPII promoter 1 |
Sample type |
genomic |
|
|
Channel 1 |
Source name |
Ntera2
|
Organism |
Homo sapiens |
Characteristics |
ChIP sample immunoprecipitated with RNAPII antibody
|
Extracted molecule |
genomic DNA |
Extraction protocol |
ChIP assay was performed following the ChIP protocol provided at http://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 |
Ntera2
|
Organism |
Homo sapiens |
Characteristics |
Total input DNA (control)
|
Extracted molecule |
genomic DNA |
Extraction protocol |
ChIP assay was performed following the ChIP protocol provided at http://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 |
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
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Submission date |
Feb 06, 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 |
GPL5989 |
Series (1) |
GSE10504 |
Using ChIP-chip technology to reveal common principles of transcriptional repression in normal and cancer cells |
|