Blood was initially collected in sodium heparin-containing Vacutainer CPTTM cell separation tubes (Becton Dickinson, Rutherford, NJ) to separate peripheral blood mononuclear cells from other elements within 2 hours from blood draw. Subsequently, T cells were isolated with the anti-CD4 coated magnetic beads, respectively, using AutoMACs automated magnetic separation unit (Miltenyi Biotec, Bergisch Gladbach, Germany). DNA and RNA were isolated from samples simultaneously using the AllPrep DNA/RNA Mini Kit (Qiagen, Inc., Hilden, Germany).
Label
biotin
Label protocol
The Illumina TotalPrep-96 RNA Amplification Kit (Ambion/Applied Biosystems, DaeMStadt, Germany) was used for reverse transcription, and amplification with 500 ng of input total RNA (at 11ul).
Hybridization protocol
700 ng of biotinylated cRNA was hybridized to a BeadChip at 580C for 16–17 hours.
Scan protocol
Illumina Bead Array Reader
Description
human peripheral CD4+ cells
Data processing
Data corrected for local background were obtained from Illumina’s proprietary software GenomeStudio. QC analyses and bead type summarization (average bead signal for each type after outlier removal) were performed using the beadarray package (25). Detection P-values were computed using the negative controls on the array. The neqc function of the limma (26) package was used to perform a normal-exponential convolution model analysis to estimate non-negative signal, quantile normalization using all probes (gene and control, detected and not detected) and samples, addition of a recommended (small) offset, log2 transformation, and elimination of control probe data from the normalized expression matrix. Sample data table cotains quantile normalized values - neqc function of the limma package was used to perform a normal-exponential convolution model analysis to estimate the quantile normalization using all probes/samples.
