Tissuelyser magna lyser

Subcutaneous white adipose tissues were excised from mice and immediately snap-frozen in liquid nitrogen. Total RNA was isolated following tissue homogenization in Trizol (Invitrogen, Carslbad, CA) utilizing a TissueLyser (MagNA Lyser, Roche), then isolated using an RNeasy RNA extraction kit (Qiagen). The quality and quantity of the RNA was determined by absorbance at 260/280 nm cDNA was prepared by reverse transcribing 1 ug of RNA with an iScript cDNA Synthesis Kit (BioRad, Hercules CA). Results were calculated using the threshold cycle method [25] (link), with β-actin utilized for normalization. For RNA deep sequencing, total sWAT RNA (10 ng) was submitted for transcriptome sequencing (RNA-Seq). For Illumina microarray, total cDNA was synthesized from BAT and spotted onto a mouse Illumina BeadArray platform (Illumina, Inc.). Fold-changes and significance were calculated based on three independent replicates. Key gene lists and cluster analyses of the data sets were performed using Ingenuity software (Ingenuity Systems Inc.).