Primary naïve CD4+ T cells were cultured for 24 h in RPMI1640 medium supplemented with plate-coated anti-CD3 (clone 2C11, 2 μg/ml), anti-CD28 (clone 57.31, 0.5 μg/ml) antibodies, 2-Mercaptoethanol (Invitrogen, 55 μM), 10% fetal bovine serum, and further supplemented with anti-IFN-γ (clone R4-6A2, 1 μg/ml), anti-IL-4 (clone 11B11, 1 μg/ml) antibodies, IL-2 (10 ng/ml, PeproTech) and recombinant human TGF-β1 (0.2 or 0.5 ng/ml, BioLegend).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with RNAiso PLUS (TAKARA). Samples were further cleaned using an NucleoSpin RNA Clean-up XS (MACHEREY-NAGEL). RNA was quantified using a Quantus fluorometer (Promega) and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng RNA using the Low input Quick Amp labeling kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy mni kit purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
Using Gene Expression Hybridiation kit (Agilent), 600 ng of Cy3-labelled cRNA (Cy3 incorporation > 6) was fragmented at 60°C for 30 minutes following the manufacturers instructions. On completion of the fragmentation reaction, samples were hybridized to SurePrint G3 Mouse GE microarray kit 8x60k (G4852A, Agilent) for 17 hours at 65°C, rotating in hybridization oven (G2545A , Agilent) at 10 rpm. 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 (G2539A) using a setting AgilentG3_GX_1color (Scan Area 61x21.6 mm, Scan resolution 3 um, Dye channel is set to Green and Green PMT is set to 100%).
Description
Gene expression after 24 h in iTreg cell differentiation condition of Foxp3-KO naive CD4+ T cells.
Data processing
The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent) using default parameters (protocol GE１_1010_Sep10, QC metric GE１_QCMT_Sep10, and Grid: 028005_D_F_20131202) to obtain background subtracted and spatially detrended Processed Signal intensities. Output data were further analyzed with GeneSpring GX 12.6.1 with a default setting, with Prcentile=75, to generate values of normalized signal intensity.
