The cells were activated with plate bound anti-CD3 and anti-CD28 for 48 hours.
Growth protocol
Human primary CD4+ T cells were isolated from peripheral blood and cultured in IL-2 containing medium.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated with RNeasy columns (Qiagen)
Label
biotin
Label protocol
labelling was performed according to the Affymetrix (Santa Clara, CA) GeneChip Whole Transcript (WT) Sense Target Labelling Assay manual
Hybridization protocol
microarrays were hybridized overnight with 5 mg biotin labelled ss-cDNA, and washed in fluidics station wash protocol: MES_EukGE-WS2v5_450
Scan protocol
Genechip scanner 3000 7G
Description
FileNumber: 2
Data processing
The input files were normalized with full quantile normalization (Irizarry et al 2003). For each input array, for each probe expression value, the array ith percentile probe value was replaced with the average of all array ith percentile points. Next, the 6,553,590 probes were manipulated into the analysis values as follows. Probes with GC count less than 6 and greater than 17 were excluded from the analysis. Probe scores were then transformed by taking the Natural Logarithm of 0.1 plus the probe score. Background CorrectionExon arrays do not use individual mis-match probes. Background is established from a pool of probes designed for that purpose. Background probes are stratified by CG content and are defined in the Human Exon 1.0 ST_antigenomic.bgp file. BGP files can also be downloaded from www.affymetrix.com. Each probe score was corrected for background by subtracting the median expression score of background probes with similar GC content. The Human Exon 1.0 ST array contains 1,404,693 probe-sets (typically, but not always, groups of four probes). Probe-set Expression Scores and Annotation FilteringThe expression score for a probe-set was defined to be the median of its probe expression scores and probe-sets with fewer than 1 probes (that pass all of the tests defined above) are excluded from further analysis. Exon Array Probes are designed off of genomic sequence and hence the reliability of probes and probe-sets correspond to the quality of their parent genomic annotations. Probe-set reliability is ranked from more to less reliable as Core, Extended, or Full. For example 'Core' probe-sets include probe-sets that correspond to high quality genomic features like RefSeq (www.ncbi.nlm.nih.gov) or Ensembl (www.ensembl.org) transcripts while 'full' and 'extended' probe-sets match less reliable annotations like EST hits and gene prediction algorithms. For this analysis, only 'Core' probe-sets were analyzed.
