Two to 4 independent hypothalamic nuclei punches were pooled together per biological replicate. Hypothalamic punches were homogenized manually using a Pellet-Pestle (Kimble, 749515) for 1 minute with 1 mL QIAzol Lysis Reagent (QIAGEN). Total RNA was purified and collected from RNeasy Mini spin columns according to the RNeasy Lipid Mini Kit (QIAGEN, 74804). Total RNA was isolated from snap-frozen brown adipose tissue, WAT, and liver, which were mechanically homogenized in TRIzol (Life Technologies) using a TissueLyser (QIAGEN) for 2 minutes. Following RNA extraction with chloroform, mRNA was further purified with RNeasy Mini spin columns and on-column DNase digestion (QIAGEN).

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