NIH 3T3 cells were treated with 200 nM RheoSwitch Ligand (RSL-1) purchased from New England Biolabs (Ipswich, MA, USA). Cells were harvested 24 hours after the RSL-1 treatment.
Growth protocol
7500/cm2 of NIH 3T3 cells were seeded on 14.5 cm petri dish and cultivated overnight in DMEM containing 4.5 g/l glucose (Gibco, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum (Sigma Aldrich, St. Louis, MO, USA), 5% penicillin/streptomycin sulfate (Gibco, Carlsbad, CA, USA), G418 (500 mg/ml; Roche Applied Science, Penzberg, Germany) and hygromycin (500 mg/ml; Merck, Darmstadt, Germany). Cells were kept at 37°C under 5% CO2 atmosphere and 95% humidity.
Extracted molecule
total RNA
Extraction protocol
Trizol extraction (Thermo Fisher Scientific, Waltham, MA, USA) of total RNA was performed according to the manufacturer's instructions.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol.
Hybridization protocol
Hybridization (16h x 45°C) was processed according to the standard Affymetrix protocol.
Scan protocol
Affymetrix GeneArray Scanner3000
Description
Cells harvested after RSL1 treatment
Data processing
The data were analyzed with a commercial software called JMP Genomics, version 4, from SAS. Gene expression profiling was performed using arrays of Mouse430_2-type from Affymetrix. A Custom CDF Version 10 with Entrez based gene definitions was used to annotate the arrays. The Raw fluorescence intensity values were normalized applying quantile normalization, RMA background correction and Medianpolish Probeset Summary
