GSM6415175: high fat diet fed - SVF-HFD-2 paired-end; Mus musculus; RNA-Seq
Adipocytes and stromal vascular cells (SVCs) were prepared from adipose tissue. Briefly, epididymal fat pads were weighed, rinsed three times in PBS, and then minced in FACS buffer (PBS + 1% low endotoxin BSA). Tissue suspensions were treated with collagenase (1 mg/ml, Sigma-Aldrich) for 30 min, and then were filtered through a 100 μm filter (BD Biosciences). After centrifugation at 500 g for 5 min, the supernatant containing adipocytes was removed and the pallet containing SVCs fraction was incubated with RBC lysis buffer (eBioscience) for 5 min followed by another centrifugation (300 g, 5 min) and resuspension in FACS buffer. Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer's instructions. The quality of the RNA was assessed using the Tapestation 2200 (Agilent) and all samples passed an RNA Integrity score (RIN) above 7.5 denoting good quality RNA. Libraries were prepared using TruSeq Library prep kits (Illumina) and run on the Hiseq 2500 (Illumina) to obtain approximate coverage of 10 million reads per sample