Log 2 transformed expression signals were normalized separately per tissue as follows: quantile normalization was performed across the 3 replicates of each individual followed by quantile normalization across all individuals. VALUE = Log2 transformed normalized intensities
The arrays were scanned on the BeadArray Reader following the standard Illumina protocol. Preliminary data analysis and QC was carried out using the BeadStudio software (Illumina).
Hybridization of labeled cRNA to the BeadChip was performed according to the standard Illumina hybridization protocol.
The three technical replicates were made from the same biological sample as follows: for each single extraction per sample there were 2 in-vitro transcriptions made, the 2 in-vitro transcriptions were then pooled to make a 3rd in-vitro transcription and then all 3 were hybridized separately.
Biotinylated cRNA was prepared through two one-quarter scale Message Amp II reactions (Ambion, Foster City, CA).
Punch biopsies (8mm) were taken from a photo-protected area adjacent and inferior to the umbilicus. Subcutaneous adipose tissue was dissected from each biopsy, weighted and immediately stored in liquid nitrogen. Similarly, the remaining skin tissue was weighted and stored in liquid nitrogen. Peripheral blood samples were collected and lymphoblastoid cell lines (LCLs) were generated by Epstein Barr Virus transformation of the B-lymphocyte component by the European Collection of Cell Cultures agency.
Total RNA was extracted using the TRI Reagent procedure (Applied Biosystems/Ambion), quantified with NanoDrop (Thermo Scientific, Waltham, MA) and analyzed with a 2100 Bioanalyzer (Agilent, Santa Clara, CA).