RNA-seq of adipose tissue transcriptome

For RNA-seq, RNA was isolated from gWAT as just described. RNA was prepared for RNA-seq as described previously [8 (link)]. Briefly, RNA with RIN > 8 was subjected to poly-dT pulldown using magnetic beads (NEB) before preparation for RNA-seq using RNA Ultra kits (NEB). Libraries were sequenced on a NextSeq 500 (Illumina), and reads were aligned to the mm10 transcriptome using HISAT2 [9 (link)] after adaptor trimming using cutadapt [10 ]. Read counts per gene for RefSeq genes were computed using featureCounts [11 (link)]. Counts were normalized to reads per kilobase per million (RPKM) and processed for pairwise differential expression analysis of selected conditions using DESeq2 [12 (link)] with a false discovery rate (FDR)-adjusted p value cutoff of 0.05. Gene ontology analysis was performed using the PANTHER database [13 (link)].