Cells were activated on 24-well plates, 4x10^6 cells/ml Yssel´s medium, through the T cell receptor (plate bound antiCD3 500 ng/well, and soluble antiCD28 500 ng/ml, Immunotech, France). To induce Th1 polarization, IL-12 (2.5ng/ml, R&D Systems) was added. To induce Th2 polarization, both IL-4 (10ng/ml, R&D Systems) and anti-IL-12 (10µg/ml, R&D Systems) were added. At 48h timepoint IL-2 (17ng/ml, R&D Systems) was added to the culturing medium. Cells were harvested at 0.5, 1, 2, 4, 6, 12, 24, 48 and 72 h. Naive cells without any treatments were collected as 0h controls from 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 manufacturers instructions.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with RNeasy mini kit (Qiagen) according to manufacturers 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 was started from 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 45*C. 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 raw microarray data were processed using robust multi-array average normalization and log2-transformed in R (version 2.12.0) using the Bioconductor affy package (version 1.28.0)
An integrative computational systems biology approach identifies differentially regulated dynamic transcriptome signatures which drive the initiation of human T helper cell differentiation
