14 protocols
AccessionNameType
 
P-GSE40624-1
bioassay_data_transformation
ID_REF = VALUE = Expression values were converted from log 2.
P-GSE40624-7
image_aquisition
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
P-GSE40624-8
feature_extraction
The raw data in the .CEL files were normalized by the GCRMA method (GC robust multi-array analysis). It is implemented in Bioconductor (http://www.bioconductor.org/). The raw signal intensity data were normalized, background corrected and summarized based on certain statistical models, and an expression value, in log2-scale, is obtained per chip per probe set.
P-GSE40624-6
hybridization
Hybridization, etc was performed by the BCM Microarray Core Facility in Houston, TX.
P-GSE40624-5
labeling
Biotinylated cRNA were prepared according to the standard Affymetrix protocol in the Baylor College of Medicine Microarray Core Facility, Houston, TX.
P-GSE40624-12
image_aquisition
Arrays were scanned on an Affymetrix Scanner 3000 7G
P-GSE40624-13
feature_extraction
Data was analyzed using MAT at http://liulab.dfci.harvard.edu/MAT/ CplusInp.bed.txt contains all 1581 high quality STAT5 binding sites.
P-GSE40624-11
hybridization
Approximately 8.25μg of DNA was hybridzed per array using the Affymetrix hybridization kit at the EMBL Genomics Core Facility in Heidelberg, Germany.
P-GSE40624-10
labeling
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.
P-GSE40624-3
grow
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).
P-GSE40624-2
specified_biomaterial_action
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.
P-GSE40624-4
nucleic_acid_extraction
Extraction of total RNA was performed using Qiagen Rneasy Kit according to the manufacturer's instructions.
P-GSE40624-9
nucleic_acid_extraction
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.