RNA-Sequencing Library Preparation

Frozen liver tissue was homogenized in 1 ml of TRIzol Reagent (Ambion by Life Technologies) using a powered Qiagen TissueLyser II homogenizer. Liver RNA was isolated using the protocol accompanying the TRIzol Reagent; after the “RNA resuspension” step, the total RNA was treated with DNase and purified (DNA-free Kit, Ambion by Life Technologies, AM1906). Frozen adipose and hypothalamus samples were processed using the Qiagen RNeasy Lipid Tissue Mini Kit (74804). Additionally, The RNase-Free DNase Set (Qiagen, 79254) was used to treat each adipose and hypothalamus sample. Highest quality RNA samples were selected. Selected RNA samples were verified to have an A260/A280 ratio between 1.8 and 2.1 on a NanoDrop UV–Vis Spectrophotometer (Thermo Scientific). Additionally, sample integrity was verified using a fragment analyzer (RNA quality number: M = 8.5, SD = 0.85). Subsequently, 1 μg of DNase-treated and purified total RNA from each sample was used for mRNA enrichment and RNA-seq library preparation.

Paired-end, 50-base pair RNA-seq libraries were generated on an Illumina HiSeq 2500 platform (using the Rapid Run mode) in the University of Wisconsin-Madison Biotechnology Center. RNA-seq library preparation and construction included the use of Illumina TruSeq RNA Sample Prep kits (v2, sets A and B; RS-122-2001 and RS-122-2202), which enabled the generation of mRNA-enriched libraries from the total RNA, and Illumina TruSeq Rapid Cluster-HS kits (PE-402-4001), which enabled the formation of clonal template clusters during the HiSeq 2500 runs, and Illumina TruSeq Rapid SBS kits (FC-402-4002) for the sequencing of reads.