Rneasy universal midi kit

EAT was homogenized and RNA was isolated using RNeasy Universal Midi kit (Qiagen, Valencia, CA, USA) per the manufacturer’s instructions for lipid rich tissue. Isolated RNA was treated with Turbo DNase (Ambion, Waltham, MA) to minimize genomic DNA contamination. RNA quality was assessed using the Experion RNA analysis electrophoresis kit (Bio-Rad, Hercules, CA, USA). Only samples with an RNA Quality Indicator (RQI) greater than 7 were sequenced.

Gene expression of EAT homogenates was determined by RNA sequencing as described previously [23 (link),24 (link)]. EAT RNA was isolated using RNeasy Universal Midi kit (Qiagen, Valencia, CA) per the manufacturer’s instructions. RNA was treated with Turbo DNase (Ambion, Waltham, MA) and RNA quality was assessed using an Experion RNA analysis electrophoresis kit (Bio-Rad, Hercules, CA). Only samples with an RNA Quality Indicator (RQI) greater than 7 were sequenced. Samples were prepared for sequencing using the Illumina TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA) and AMPure XP beads (Beckman Coulter, Brea, CA) per the manufacturer’s instructions. DNA fragment size was determined using Experion DNA 1 K chips (Bio-Rad, Hercules, CA) and libraries were quantified using the KAPA Library Quantification kit (KAPA Biosystems, Wilmington, MA). Samples were sequenced using an Illumina NextSeq 500 sequencer (Illumina, San Diego, CA) with 100 base pair single end reads. Raw FASTQ data were processed for quality using CLC Bio Genomic Workbench (Qiagen, Valencia, CA). The EAT transcriptome was assembled using the annotated Sus scrofa 11.1-build as a reference genome [25 ]. Additional analysis was also performed using the Porcine Translational Research Database [26 ,27 (link)], as described below.