Deoxyribonuclease treatment

Total RNA was extracted using an RNeasy mini kit (Qiagen, 74104). The RNA was subjected to deoxyribonuclease treatment (Qiagen, 79254), and its concentration was measured with a Tecan plate reader. Complementary DNAs (cDNAs) were synthesized using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, 4368814) according to the manufacturer’s protocol. qRT-PCR was performed in a 10-μl reaction containing 10 ng of cDNA, 5 μl of Applied Biosystems PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, A25741), and 0.4 μl of each 10 μM forward and reverse primer for the target genes. Pgk was used as a housekeeping gene. The sequence of the primers for each target gene is described in table S1. For Pax7, Myf5, and Myog presented in Fig. 1C and fig. S1B, probe-based qPCR was conducted according to the manufacturer’s protocol using PrimeTime qPCR assays purchased from Integrated DNA Technologies (Coralville, USA); mGapdh (Mm.PT.39a.1), mPax7 (Mm.PT.58.12398641), mMyf5 (Mm.PT.58.5271235), and mMyog (Mm.PT.58.30712483.gs).

All rat or mouse brains were rapidly extracted after light (4%) isoflurane (Baxter AB, Sweden) anesthesia, quickly frozen in isopentane (on dry ice chamber) and transferred to −80°C freezer; PBN, hypothalamus, amygdala, and hippocampus were collected. Note that while for rat brain dissections lPBN was reliably dissected, in mice the dissections likely encompassed lateral and dorsal parts of the mPBN. Total RNA was extracted using RNeasy Lipid Tissue Mini kit (QIAGEN) with additional deoxy ribonuclease treatment (QIAGEN), or using PicoPure™ RNA isolation kit (Thermofisher). RNA quality and quantity were assessed spectrophotometrically by Nanodrop 1000 (NanoDrop Technologies). cDNA was synthesized using iScript cDNA Synthesis kit from Bio-Rad. Real-time RT-PCR was performed using TaqMan probes, and selective primer sets described in the key resource table.
Gene expression values were calculated based on the ΔΔCt method (Livak and Schmittgen, 2001 (link)). For initial assessment of gene expression changes in mice each group consisted of 10 mice (per diet and per sex). For rats: 5 males were included in each diet group, and 11 females were included in the chow group and 12 in the HFSD group.