|
| Status |
Public on Nov 06, 2011 |
| Title |
Jurkat Tat cells vs Jurkat cells H3K9ac ChIP-chip |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Jurkat-Tat
|
| Organism |
Homo sapiens |
| Characteristics |
expression: HIV Tat protein
chip antibody: H3K9ac
cell line: Jurkat
chip antibody manufacturer: Upstate
chip antibody catalog #: 07-352
chip antibody manufacturer: Upstate
chip antibody catalog #: 07-352
|
| Growth protocol |
Jurkat-Tat cells were maintained in RPMI supplemented with 10% fetal bovine serum, 1%penicillin and streptomycin and 800 microg/ml of G418. Jurkat T cells were obtained from ATCC and cultured under standard tissue culture conditions.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
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 were used for immunoprecipitation with anti-Histone H3 acetyl Lys9 antibody; 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 DNA were amplified using the WGA Kit (Sigma)(3).
|
| 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 IP from Jurkat cells and IP from Jurkat-Tat 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 |
Jurkat
|
| Organism |
Homo sapiens |
| Characteristics |
expression: control
chip antibody: H3K9ac
cell line: Jurkat
|
| Growth protocol |
Jurkat-Tat cells were maintained in RPMI supplemented with 10% fetal bovine serum, 1%penicillin and streptomycin and 800 microg/ml of G418. Jurkat T cells were obtained from ATCC and cultured under standard tissue culture conditions.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
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 were used for immunoprecipitation with anti-Histone H3 acetyl Lys9 antibody; 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 DNA were amplified using the WGA Kit (Sigma)(3).
|
| 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 IP from Jurkat cells and IP from Jurkat-Tat 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 ChIP-on-chip microarray (244K) (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.
|
| Data processing |
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
|
| |
|
| Submission date |
Jul 18, 2011 |
| Last update date |
Nov 06, 2011 |
| Contact name |
Siavash K Kurdistani |
| E-mail(s) |
Skurdistani@mednet.ucla.edu
|
| Phone |
310-794-5194
|
| Fax |
310 206-5272
|
| Organization name |
University of California Los Angeles
|
| Department |
Biological Chemistry
|
| Street address |
615 Charles E. Young Dr. South, 337
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
| |
|
| Platform ID |
GPL7032 |
| Series (2) |
| GSE30736 |
Genome-wide binding map of the HIV Tat protein to the human genome (ChIP-chip) |
| GSE30739 |
Genome-wide binding map of the HIV Tat protein to the human genome |
|
