|
| Status |
Public on Aug 26, 2008 |
| Title |
ChIP on chip R2G e1a (IP infected versus INP infected) at 24 hours post infection |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
IP Infected IMR90 fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
IP Infected IMR90 fibroblasts
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
2x107 IMR90 lung fibroblasts were grown on 10 cm dishes to confluence. After 24 h, the cells were incubated with mock- or the dl1500 adenovirus for 1 h in low serum media (2%). At the indicated times post infection (p.i.), formaldehyde was added for 10 min at 37°C. After PBS washing, cross-linked cells were scraped from the plates and washed with 1 ml of PBS containing protease inhibitors (Roche). Cells were resuspended in 400 µl of Lysis buffer and incubated for 10 min on ice and immediately sonicated. 100 µl of the lysate (corresponding to 5x106 cells) were used for immunoprecipitation with a given antibody (listed in table S1); 10 µl of the lysate was used as input. After overnight reversal of crosslinking at 65°C, samples were treated with RNase A for 30 min at 37°C and subsequently purified using the Qiagen Qiaquick PCR purification Kit. 10 ng of each IP and INP DNA were amplified using the WGA Kit (Sigma)
|
| Label |
cy5
|
| Label protocol |
2 μg of amplified material were labeled with Cy3 or Cy5 (PerkinElmer) using the Bioprime Labeling Kit (Invitrogen). DNA was mixed with 35 µl of random priming solution (Invitrogen Bioprime Kit) to a final volume of 75 µl, boiled for 5 min and quickly cooled in an ice-water bath for 5 min. The labeling reaction was completed with 60U Klenow, dNTPs (0.12 mM dATP, dGTP and dTTP and 0.06 mM dCTP), 1.28 mM Cy3 and Cy5 for input and IP (or IP from mock and IP from dl1500-infected cells) respectively, and incubated for 3h at 37°C. The labeled DNA was purified using Qiagen Qiaquick PCR purification Kit and the incorporation was measured with Nanodrop.
|
| |
|
| Channel 2 |
| Source name |
INP Infected IMR90 fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
INP Infected IMR90 fibroblasts
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
2x107 IMR90 lung fibroblasts were grown on 10 cm dishes to confluence. After 24 h, the cells were incubated with mock- or the dl1500 adenovirus for 1 h in low serum media (2%). At the indicated times post infection (p.i.), formaldehyde was added for 10 min at 37°C. After PBS washing, cross-linked cells were scraped from the plates and washed with 1 ml of PBS containing protease inhibitors (Roche). Cells were resuspended in 400 µl of Lysis buffer and incubated for 10 min on ice and immediately sonicated. 100 µl of the lysate (corresponding to 5x106 cells) were used for immunoprecipitation with a given antibody (listed in table S1); 10 µl of the lysate was used as input. After overnight reversal of crosslinking at 65°C, samples were treated with RNase A for 30 min at 37°C and subsequently purified using the Qiagen Qiaquick PCR purification Kit. 10 ng of each IP and INP DNA were amplified using the WGA Kit (Sigma)
|
| Label |
cy3
|
| Label protocol |
2 μg of amplified material were labeled with Cy3 or Cy5 (PerkinElmer) using the Bioprime Labeling Kit (Invitrogen). DNA was mixed with 35 µl of random priming solution (Invitrogen Bioprime Kit) to a final volume of 75 µl, boiled for 5 min and quickly cooled in an ice-water bath for 5 min. The labeling reaction was completed with 60U Klenow, dNTPs (0.12 mM dATP, dGTP and dTTP and 0.06 mM dCTP), 1.28 mM Cy3 and Cy5 for input and IP (or IP from mock and IP from dl1500-infected cells) respectively, and incubated for 3h at 37°C. The labeled DNA was purified using Qiagen Qiaquick PCR purification Kit and the incorporation was measured with Nanodrop.
|
| |
|
| |
| Hybridization protocol |
Hybridization onto the Human Promoter array (Agilent-G4489A), washing, and scanning were carried out according to the manufacturer's instructions
|
| Scan protocol |
The arrays were scanned using an Agilent DNA Microarray scanner.
|
| Description |
Data extraction and analyses were carried out using the Agilent Feature Extraction software (version 9.1.3.1) and Chip Analytics software (version 1.2). Probe signals were extracted with the Agilent Feature extraction software, normalized with Lowess normalization using the Chip Analytics software
|
| Data processing |
Each ChIP profile was normalized to generate mean value of zero and variance of one, since the assay accurately captures the relative enrichment of an IP across an array.
|
| |
|
| Submission date |
Jul 09, 2008 |
| Last update date |
Aug 25, 2008 |
| Contact name |
Siavash K Kurdistani |
| E-mail(s) |
Skurdistani@mednet.ucla.edu
|
| Organization name |
UCLA
|
| Department |
Biological Chemistry
|
| Lab |
Kurdistani
|
| Street address |
615 Charles E Young Dr South
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
| |
|
| Platform ID |
GPL7032 |
| Series (2) |
| GSE12046 |
Expression profiles and ChIP on chip genomewide experiments with R2G mutant virus |
| GSE12543 |
Profiles and ChIP on chip experiments with dl1500 virus (expressing WT small e1a), R2G and deltaCR2 mutant viruses |
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