Human primary Th1, Th2, Th17 and Treg cells were purified by flow cytometry sorting. Total RNA was extracted from naïve CD4+ T cells (NT), CD4+ T central memory T cells (TCM), Th1, Th2, Th17 and Treg subsets using a miRneasy Mini Kit (Qiagen, Valencia, CA, USA).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 45ng total RNA using a LowInput QuickAmp Labeling Kit (Agilent Technologies, Palo Alto, Calif, USA) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
600 ng of Cy3-labelled cRNA (specific activity >6 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a total volume of 25ul following the manufacturers instructions. On completion of the fragmentation reaction, 25ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4851A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner using one color scan setting for 8x60k array slides.
Data processing
The scanned images were analyzed with Feature Extraction Software (Agilent). Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
