FACS sorting All antibodies used for cell sorting were purchased from BD Pharmingen (San Diego, CA). Thymocytes were harvested from 5- to 6-week-old female C57BL/6 mice. Four independent cell sortings were performed and four mice were sacrificed for each experiment. Before cell sorting, CD4+ cells and CD8+ cells were depleted using the MACS LD system (Miltenyi Biotec, Bergisch Gladbach, Germany). The remaining fraction was stained with anti-CD44 and anti-CD25 antibodies conjugated to FITC or PerCP-Cy5.5, respectively, and also with PE-conjugated antibodies to CD4, CD8, CD3, NK1.1, and TCRgamma-delta and sorted using a FACSAria cell sorter (BD Bioscience). DN2, DN3, and DN4 subsets were identified as FITC+PE-PerCP-Cy5.5+, FITC-PE-PerCP-Cy5.5+, FITC-PE-PerCP-Cy5.5- populations, respectively.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from sorted cells using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction.
Label
biotin
Label protocol
Biotin-labeled cRNA probes were prepared using Two-Cycle cDNA synthesis kit® (Affymetrix®, Santa Clara, CA), according to Affymetrix protocol.
Hybridization protocol
Following fragmentation, Biotin-labeled cRNA was hybridized to the Mouse Genome 430 2.0 Array® (Affymetrix®) for 16 h at 45°C as recommended by the manufacturer. Washing was performed using an automated fluidics workstation, and the array was immediately scanned with GeneChip Scanner 3000 7G.
Scan protocol
The array was scanned with GeneChip Scanner 3000 7G.
Description
Cell source of DN2_1, DN3_1, and DN4_1 are identical. Cell source of DN2_2, DN3_2, and DN4_2 are identical. Cell source of DN2_3, DN3_3, and DN4_3 are identical. Cell source of DN2_4, DN3_4, and DN4_4 are identical. Each cell sorce was derived from four mice.
Data processing
Expression data were extracted from image files produced on Affymetrix® GeneChip Operating Software 1.0® (GCOS®).
