|
| Status |
Public on Mar 21, 2013 |
| Title |
Cplus vs Input |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
STAT5 ChIP DNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: leukemia cell
cell line: Kit225
antibody: STAT5 C-term polyclonal
|
| Treatment protocol |
Prior to IL-2 stimulation, Kit225 cells were made quiescent for 24 h in their regular medium without IL-2. Cell stimulations were carried out with 10 nM IL-2.
|
| Growth protocol |
Cells were maintained in RPMI-1640 medium containing 10% fetal calf serum, 2 mM L-glutamine and penicillin-streptomycin (50 IU/ml and 50 μg/ml, respectively). Kit225 media was supplemented with 20 U/ml human recombinant IL-2 (NCI Preclinical Repository).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitations were performed from approximately 5 x 107 Kit225 cells as described by our group earlier (Nagy et al Mol. Cancer. 2009) with anti-STAT5A/B antibodies (mixing sc-1081 and sc-835 (C-terminal STAT5A and STAT5B antibodies, respectively)) or normal rabbit serum (IgG control) for 3h at 4 ºC. To confirm that the assays were successful, PCR amplification of the known STAT5 binding site located 5’ to the human IL2RA gene within the Positive Regulatory Region III (Forward: 5’-ACG TCT AGA AAG AAA GTG GTC-3’ Reverse: 5’- CTG TCC CTG GAT GAA CCT AGT-3’) was performed using quantitative real time PCR.
|
| Label |
biotin
|
| Label protocol |
To generate libraries from ChIP-ed DNA, random amplification was carried out as described at http://research.stowers-institute.org/gertonlab/protocols/RandomPCRamplification.pdf by the DeRisi lab at the UC San Francisco.
|
| |
|
| Channel 2 |
| Source name |
Input DNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: leukemia cell
cell line: Kit225
antibody: input, none
|
| Treatment protocol |
Prior to IL-2 stimulation, Kit225 cells were made quiescent for 24 h in their regular medium without IL-2. Cell stimulations were carried out with 10 nM IL-2.
|
| Growth protocol |
Cells were maintained in RPMI-1640 medium containing 10% fetal calf serum, 2 mM L-glutamine and penicillin-streptomycin (50 IU/ml and 50 μg/ml, respectively). Kit225 media was supplemented with 20 U/ml human recombinant IL-2 (NCI Preclinical Repository).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitations were performed from approximately 5 x 107 Kit225 cells as described by our group earlier (Nagy et al Mol. Cancer. 2009) with anti-STAT5A/B antibodies (mixing sc-1081 and sc-835 (C-terminal STAT5A and STAT5B antibodies, respectively)) or normal rabbit serum (IgG control) for 3h at 4 ºC. To confirm that the assays were successful, PCR amplification of the known STAT5 binding site located 5’ to the human IL2RA gene within the Positive Regulatory Region III (Forward: 5’-ACG TCT AGA AAG AAA GTG GTC-3’ Reverse: 5’- CTG TCC CTG GAT GAA CCT AGT-3’) was performed using quantitative real time PCR.
|
| Label |
biotin
|
| Label protocol |
To generate libraries from ChIP-ed DNA, random amplification was carried out as described at http://research.stowers-institute.org/gertonlab/protocols/RandomPCRamplification.pdf by the DeRisi lab at the UC San Francisco.
|
| |
|
| |
| Hybridization protocol |
Approximately 8.25μg of DNA was hybridzed per array using the Affymetrix hybridization kit at the EMBL Genomics Core Facility in Heidelberg, Germany.
|
| Scan protocol |
Arrays were scanned on an Affymetrix Scanner 3000 7G
|
| Description |
Technical replicates
|
| Data processing |
Data was analyzed using MAT at http://liulab.dfci.harvard.edu/MAT/
CplusInp.bed.txt contains all 1581 high quality STAT5 binding sites.
|
| |
|
| Submission date |
Sep 05, 2012 |
| Last update date |
Mar 21, 2013 |
| Contact name |
Zsuzsanna S. Nagy |
| Organization name |
University of Debrecen
|
| Department |
Biochemistry and Molecular Biology
|
| Street address |
Nagyerdei krt. 98
|
| City |
Debrecen |
| ZIP/Postal code |
H-4032 |
| Country |
Hungary |
| |
|
| Platform ID |
GPL5082 |
| Series (3) |
| GSE40618 |
IL-2 induced gene expression changes in Kit225 human leukemia cell lines |
| GSE40619 |
Genome-Wide STAT5 binding sites in Kit225 leukemia cells |
| GSE40624 |
Genome-wide mapping of IL-2 regulated target genes and IL-2 activated STAT5 binding sites in Kit225 human leukemia cells. |
|
