Expression profiles of serotonergic receptor genes in ARC or AVPV kisspeptin neurons were analyzed using RNA-seq data reported previously67 (link). Briefly, the ARC or AVPV tissues were dissected out from Kiss1-tdTomato heterozygous female rats under OVX (n = 2) or OVX + high E2 (n = 2) conditions, respectively, because tdTomato fluorescence expression was dependent on Kiss1 expression and the tdTomato signals were only detectable in the ARC of OVX rats and the AVPV in OVX + high E2 model rats9 (link). The ARC or AVPV tissues were minced and treated with papain and then gently dispersed by repetitive pipetting, and the tdTomato-positive cells were picked up under a fluorescent microscope by pipettes (inner diameter: 20–30 μm)71 (link). Ten (for ARC) or 3 (for AVPV) tdTomato-positive cells were pooled into a polymerase chain reaction tube containing an RNase inhibitor (RNasin Plus; Promega, Madison, WI, USA). Gene expression levels were normalized by the reads per kilobase million mapped reads for each mRNA67 (link).
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