For RNA isolation from each fat depot from 5 animals within each nutritional group, and a 100 mg was used from samples collected at 7, and 1000 mg from taken at 28 days of age, respectively. These were mixed with 2 ml of TRI reagent (Sigma-Aldrich). Total RNA was extracted using the RNeasy Plus kit (Qiagen) according to the manufacturer’s instructions and its quantity measured with a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific). Optical density ratios (260/280 nm) were >1.9 for all samples. Total RNA quality was assayed by the Agilent BioAnalyzer RNA 6000 Nano Kit (Agilent Technologies) and only used if distinct ribosomal peaks measured (i.e. RIN > 7).
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