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The RNA-seq results of Glp1r and Lepr samples were processed using the nf-core/rnaseq pipeline (V.1.4.2)60. This involves (1) aligning the raw reads to the reference genome GRCm38.p6 using STAR (2.6.1d)61 and (2) transcript abundance estimation using Salmon (0.14.1)62 with the reference transcriptome from Ensembl (release 97)63. To normalize the ribosomal pulldown (IP) to the hypothalamic background (input) per sample, we calculated a ratio of the Salmon gene counts (IP/input). The differentially expressed genes between Glp1r and Lepr groups were derived using DESeq2 (V.1.26.0)64 where P values attained by the Wald test were corrected for multiple testing using the Benjamini–Hochberg method. This yielded genes that are regulated between the Glp1r and Lepr conditions and act as markers for these POMC populations.

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