Cells were activated on 24-well plates, 4x106 cells/1ml Yssel´s medium, through the T cell receptor (plate bound antiCD3 500 ng/well, and soluble antiCD28 500 ng/ml, Immunotech, France), and to half of the samples IL-4 (10ng/ml, R&D Systems) and anti IL-12 (10µg/ml, R&D Systems) were added to induce Th2 polarization. At 48h timepoint IL-2 (17ng/ml, R&D Systems) was added to the culture medium. Cells were harvested at 0.5, 1, 2, 4, 6, 12, 24, 48 and 72 h. Naive cells without any treatment were collected as 0h controls for every replicate.
Growth protocol
Umbilical cord blood was collected from healthy neonates born in Turku University Hospital, Hospital District of Southwest Finland. Mononuclear cells were isolated with Ficoll-Paque (Amersham Biosciences) gradient centrifugation, and CD4+ cell population was purified using magnetic beads (Dynal CD4 Positive Isolation Kit, Invitrogen) according to manufacturer's instructions.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the RNeasy mini kit (Qiagen) according to manufacturer's instructions. In-column DNase (RNase-Free Dnase Set, Qiagen) treatment was performed for 15 minutes. RNA was eluted in dH2O.
Label
biotin
Label protocol
Amplification used 100 ng of total RNA using the Affymetrix Two-Cycle cDNA Synthesis Kit (P/N 900432), and cDNA/cRNA synthesis reactions and sample cleanup steps were performed according to Affymetrix´s GeneChip Expression Analysis Technical Manual.
Hybridization protocol
15 ug of biotinylated and fragmented cRNA was hybridized to Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays overnight (16-18 hours) at 45C. GeneChips were washed and stained in the Affymetrix Fluidics Station.
Scan protocol
GeneChips were scanned using the GeneChip Scanner 3000 with AutoLoader.
Description
biological replicate 3
Data processing
The PM probe intensities were quantile-normalized using the R/Bioconductor package affy and log2-transformed. To identify consistent changes across the biological replicates, the probe-level expression change averaging procedure (PECA) was applied (Elo et al., Nucleic Acids Res 33:e193, 2005). The benefit of this approach is that is that the reliability of estimation can be improved by considering the distribution of probe-level expression changes instead of a single probeset-level value. Specifically, linear modeling with moderated F- and t-statistics was first applied to the probe-level time series data using the R/Bioconductor package limma and the obtained probe-level estimates were then summarized into probeset-level values using the Tukey biweight average. The probe-level estimates are contained in the sample (57) data tables. Since the PECA-estimation requires always at least two samples to be compared, the probeset-level data is represented as a supplemental file on the Series record and contains the PECA-estimates for the signal log-ratios between each non-Thp sample (54) and the corresponding Th cell precursor-samples (0h timepoint).
