Cells were treated with 100nM 1,25(OH)2D3 or ethanol vehicle for 24 hours.
MC3T3-E1 cells were cultured in alpha MEM with 10% heat-inactivated fetal bovine serum from Hyclone (Logan, UT) and 1% penicillin-streptomycin. Differentiated cells were cultured in complete media plus 10mM glycerol-2-phosphate and 50ug/mL ascorbic acid for 15 days, changing media every 2-3 days.
RNA was isolated using the TRI-Reagent protocol (MRC, Cincinnati, OH) and amplification methods according Roche NimbleGen protocols.
Labeling was performed using the NimbleGen Systems Inc., Madison, WI USA, standard operating protocol. See www.nimblegen.com.
Hybridization was performed by NimbleGen Systems Inc., Madison, WI, USA following their standard operating protocol. See www.nimblegen.com.
Scanning was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
The raw data (.pair file) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction as implemented in the NimbleScan software package, version 2.4.27 (Roche NimbleGen, Inc.).