To induce Meis1 deletion, 3-week-old K14-CreERT2 Meis1fl/fl (CKO) and K14-CreERT2 Meis1f//+ mice (controls) were injected intraperitoneally on 3 consecutive days with 1 mg of tamoxifen (Final concentration 20 mg/ml, Sigma-Aldrich, St Louis, MO) that was prepared following the manufacturer’s instructions by completely dissolving Tamoxifen into 200 ul of 100% ethanol at 55°C, adding 1800 ul of warmed sunflower oil, and mixing them well by vortexing. Four days after tamoxifen treatment, CD45- EpCAM+ mouse thymic epithelial cells were sorted with a FACSAreaTMII (BD Bioscience, San Diego, CA).
Extracted molecule
total RNA
Extraction protocol
Thymic fragments were stirred gently in RPMI-1640 (Wako Pure Chemical Industries) medium for 15 min at 4°C to remove free thymocytes and then transferred to fresh medium containing 1.25 mg/ml collagenase / dispase with 0.01 mg/ml DNase I (Roche Diagnostics, Basel, Switzerland), and incubated for 15 min at 37°C and subjected to vigorous pipetting. The last three steps were repeated twice, discarding the supernatant each time, and incubation was continued until tissue digestion was complete. Released cells were filtered to remove clumps. For enrichment of the TEC populations, an immunomagnetic separation technique was performed using anti-CD45 coated beads and anti-Ter119 coated beads (Miltenyi Biotec) to deplete hematopoietic cells and erythroblasts. Purified TECs were sorted with a FACSAreaTMII or FACSvantage (BD Bioscience, San Diego, CA). RNA was isolated using the Qiagen RNeasy micro kit (Qiagen) following the manufacturer's recommendations. The protocol includes differential lysis of cells, and an on-column DNase digestion. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cDNA was prepared from 10ng RNA using the WT-Ovation™ Pico RNA Amplification System (NuGEN) and Genomic Enzymatic Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cDNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
3 ug of Cy3 labeled cDNA (specific activity >15.0 pmol Cy3/ug cDNA) was hybridized to Agilent Whole Mouse Genome Oligo Microarrays (G4122F) 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 (G2505B) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
Description
gene expression in mouse thymic epithelial cells in the absence of Meis1
Data processing
The scanned images were analyzed with Feature Extraction Software 9.5.3.1 (Agilent) using default parameters (protocol: GE1-v5_95_Feb07 and Grid: 014868_D_F_20100123) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Normalized data were analyzed using GeneSpring GX version 11.0.2 software (Agilent Technologies, Santa Clara, CA). Threshold raw signals to 1.0, Normalization algorithm: Percentile Shift, Normalization: Shift to 50 percentile
