At day 11 post induction the media on the cells was replaced with DMEM supplemented with 10% FBS, 100 nM insulin, 50 units/ml penicillin, and 50 µg/ml streptomycin containing DMSO (control; n=6), GW610742 PPARd selective agonist (n=6 1uM) or GW347845 PPARg selective agonist (n=6 100uM) for 2 days prior to cell collection and RNA extraction.
Growth protocol
3T3-L1 preadipocytes were grown in T75 flasks and maintained in Dulbecco’s modified Eagles media (DMEM) (high glucose 4.5 g/l; Sigma-aldrich) supplemented with 10% (v/v) new born calf serum (Sigma-Aldrich), 50 units/ml penicillin, and 50 µg/ml streptomycin (Sigma-Aldrich) in a humidified 5% CO2 incubator at 37ºC. At 2 days post-confluence cells were induced to differentiate with DMEM supplemented with 10% v/v fetal bovine serum (FBS) (Invitrogen), 1 uM dexamethasone (Sigma-Aldrich), 0.5 mM isobutylmethylxanthine (Sigma-Aldrich), 100 nM insulin (Sigma-Aldrich), 50 units/ml penicillin, and 50 µg/ml streptomycin. The cells were maintained in this media for 72h as this was found to improve the reproducibility of differentiation between flasks. After 72 h the media was replaced with DMEM supplemented with 10% FBS, 100 nM insulin, 50 units/ml penicillin, and 50 µg/ml streptomycin. The media was subsequently changed for DMEM supplemented with 10% FBS, 50 units/ml penicillin, and 50 µg/ml streptomycin every 48 h. (Roberts et al 2009b)
Extracted molecule
total RNA
Extraction protocol
Cells were collected by removing the media and washing each T75 flask with 10 ml of PBS. Cells were then washed with 1.5 ml trypsin-EDTA solution (5 BAEE units trypsin/ml, 1.8 µg EDTA/ml; Sigma-Aldrich) for 2 min at 37ºC to remove the cells from the surface of the flask. 8.5 ml DMEM supplemented with 10% (v/v) new born calf serum, 50 units/ml penicillin, and 50 µg/ml streptomycin was added to each flask. The DMEM containing the cells was transferred to a falcon tube and centrifuged at 200 g for 2 min to pellet the cells. The remaining media was removed and the cells washed with physiological saline (0.9% NaCl) solution. RNA from 3T3-L1 adipocytes was extracted using RNeasy (Qiagen GmbH, Hilden, Germany). Approximately 5mg of cells was used per sample for RNA isolation. Procedures were carried out according to the manufacturer’s instructions. Extracted RNA was quantified and its purity assessed using a Nanodrop ND-1000 Spectrometer (Nanodrop Technologies Inc., Wilmington, USA) to measure the absorbance at 260 nm and the A260/A280 ratio, respectively.
Label
biotin
Label protocol
Standard Illumina labeling protocol
Hybridization protocol
Standard Illumina hybridization protocol
Scan protocol
Standard Illumina scanning protocol. Illumina Infinium Gene Expression BeadArrays (Illumina Inc, San Diego, CA) were used to perform transcriptomics. A mouse WG6 array platform was used with 45281 probes.
Description
replicate 6
Data processing
The R package lumi was used with the detection p-value threshold set to 0.01. Probes were required to be successfully detected (p-value<0.01 in Lumi) in at least one sample to pass the selection. The data was transformed using variance stabilization and then normalised using quantile normalisation. Gene expression was compared using the R package limma with a 5% confidence interval. Data were normalized in two batches: cont v gamm AND cont v delt. Therefore, there are two sets of normalized data for the controls (both derived from the same raw data).
