Data analysis was carried out using R (v 2.12) and BioConductor, a language for statistical computing. The data was processed using alternative Ensembl genome-based probeset definitions that create a single probeset per annotated gene. Because we have a one-to-one mapping of probesets to genes, we refer to genes rather than probesets. Variance Stabilisation Normalisation (VSN) and median polish summarisation were applied to obtain gene expression values, and differential gene regulation was assessed using limma limiting the false discovery rate (FDR) to 0.05.
array scanning protocol
Image analysis was performed using Gene Chip Operating Software (Affymetrix). Microarray Suite version 5.0 software (Affymetrix) was used to generate .dat and .cel files
Spleens were excised and homogenised, mono-nuclear cells were isolated by density gradient centrifugation, and were seeded in 24-well plates at 4*106 cells per well in RPMI 1640 supplemented with 10% foetal bovine serum. Mouse DO.11.10 splenocytes (with CD4+ T cells expressing an ova-specific T-cell receptor) were cultured in vitro in the presence of ovalbumin (Ova), IL-2, the polarising cytokines IL-12 or IL-4, in combination with alpha-IL-4 or alpha-IFN-gamma antibodies. Briefly, spleens were homogenised, mono-nuclear cells were isolated by density gradient centrifugation, and were seeded in 24-well plates at 4×106 cells per well in RPMI 1640 supplemented with 10% foetal bovine serum. Triplicate wells were cultured in the presence of Ovalbumin (Worthington, Lakewood, NJ, USA)(10 ug/ml) and several polarising cytokine cocktails: neutral (10 ng/ml IL-2), Th1 (10 ng/ml IL-2, 10 ng/ml IL-12 and 1 ug/ml a-IL-4), or Th2 (10 ng/ml IL-2, 25 ng/ml IL-4 and 1 ug/ml alpha-IFN-gamma ). Triplicate wells were subsequently harvested on days 1, 2, 3 and 4, washed with PBS and stored in TRIzol. Naive cells (4 replicates) were harvested directly after density gradient centrifugation, washed with PBS and stored in TRIzol.
The TRIzol lysates were processed according to the manufacturers instructions. Total RNA was isolated and purified using the RNeasy Micro kit (Qiagen, Hilden, Germany): 250 ul of ethanol was added to the upper aqueous phase of the processed TRIzol samples and directly transferred to the RNeasy spin columns for purification. RNA concentrations and OD 260/280 ratios were measured with the NanoDrop ND-1000 UV-VIS spectrophotometer (NanoDrop Technologies, Wilmington, USA). Assessment of total RNA quality and purity was performed with the RNA 6000 Nano assay on the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, USA).
RNA (100 ng) was labeled using the MessageAmp Premier RNA Amplification kit (Applied Biosystems)
Hybridised onto Affymetrix GeneChip Mouse Genome Arrays (430 plus 2.0, Affymetrix), according to the manufacturers recommendations.