Data matrix file was the direct outcome of the normalization algorithm. The Brainarray Ensembl R2.7 chip defintion file (CDF) was used, and as a result, the probe set identifiers in the normalised file came from the CDF file. To disambiguates the IDs, we removed the _at suffix from the Brainarray probe set identifiers, e.g. ENSMUSG00000087403_at becomes ENSMUSG00000087403 for Ensembl.
Data were normalised and 2log transformed using the gcRMA algorithm (using R2.7.1 and Bioconductor libraries)
Labeled cRNA was used for hybridization to Affymetrix Mouse GeneChip arrays (NuGO_Mm1a520177). After an automated process of washing and staining, absolute values of expression were calculated from the scanned array using the Affymetrix GCOS software
For differentiation experiments cells were seeded at 6400 cells/cm2 and were allowed to attach overnight. Growth medium was changed for differentiation medium, which includes ITS (10 ug/ml insulin, 10 ug/ml transferrin and 3 x10-8M sodium selenite) and puromycin (2 ug/ml). Differentiation medium was replaced every two days.
For each sample 2 ug high quality total RNA was labeled using the Affymetrix Eukaryotic One-Cycle Target Labeling and Control reagents to generate Biotin-labeled cRNA. The quality of the cRNA was verified using the Agilent 2100 bioanalyzer and the concentration was measured using the Nanodrop (Nanodrop Technologies, Wilmington, DE, U.S.A).
Infection of ATDC5 cells with RNAi vectors was done in the presence of 4ug/ml polybrene. Cells were incubated for 12 hours and then allowed to recover for 24 hours on fresh medium before selection pressure was applied. Infected cells were selected with puromycin (8 ug/ml) for 72 hours, before experiments were initiated; at the onset of experiments the puromycin concentration was lowered to 2 ug/ml for the duration of the experiment.
ATDC5 cells were cultured at 37 degrees C, 5% CO2, 100% humidity in DMEM/ F-12 supplemented with 5% fetal calf serum (FCS), antibiotics (100 units/ml penicillin and 100 ug/ml streptomycin), 200 mM L-glutamine on tissue culture plates (Greiner Bio-One).
Three independent replicate RNA samples were taken at 0, 2, 4, 8, 16. 24 and 72 hours post induction of differentiation from control and EGR1 knockdown cultures. Total RNA was isolated using Tri-Reagent (Sigma) according to the manufacturers' protocol. Quantity and quality of the RNA were determined by 260/280 nm and 260/230nm absorbance measurements, respectively, using the Nanodrop (Witec AG, Luzern, Switzerland). Total RNA (1 ug) for each sample/replicate was converted into first strand cDNA using the iScript™ cDNA synthesis kit (Bio-Rad, Herculus, CA, USA) according to the manufacturers' instructions.