For RNA-seq, RNA integrity was examined using Agilent High Sensitivity RNA ScreenTape. RNA samples (1 μg) with RNA integrity number greater than 7 were used for RNA cleanup and library preparation with an Illumina TruSeq Stranded Total RNA Library Prep kit according to the manufacturer’s instructions. All barcoded libraries were quantified by KAPA Library Quantification Kit (Roche), and equimolarly pooled for subsequent sequencing. All RNA-seq experiments were done with at least 3 biological replicates per condition. High-throughput sequencing data were generated through either the Functional Genomics Core at the University of Pennsylvania or Novagene. Sequencing reads were aligned to UCSC mm10 genome using STAR v 2.6 (67). Read counts were then obtained with featureCounts per the manual’s instructions (68) (Supplemental Table 2). Differentially expressed genes (cutoff defined as FDR < 0.05, transcripts per million reads [TPM] > 0.1) were identified using DESeq2 (69). Heatmaps were generated in R with package pheatmap by (i) identifying differentially expressed genes with the aforementioned cutoffs and (ii) mapping their corresponding z-transformed TPM or log2(fold change) values across all biological replicates and treatment conditions. The heatmap color scheme was scaled accordingly for optimal graphical illustrations. Gene ontology analyses and TF enrichment were performed by Enrichr webserver (32). Gene set enrichment analyses were performed with GSEA software (70) with the mouse leptin gene signaling pathway curated from WikiPathways (https://www.wikipathways.org/index.php/WikiPathways).

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