Transcriptomic Profiling of Fluorescently Labeled Kiss1 Neurons from Prepubertal (P22) and from Adult (p70-80) Female Mice of Metestrus and Proestrus Stages
LCM-Seq was performed on fluorescently labeled Kiss1-expressing neurons (n = 400 per sample) isolated from the arcuate nucleus of female mice at distinct developmental and hormonal stages: prepubertal (3 weeks old, P22; N = 3), and young adult (10 - 12 weeks old, P70 - 80) of either metestrus (N = 3) or proestrus (N = 3) phases.Experimental animals were generated by crossing male Kiss1/Cre-GFP mice (Yeo et al., 2016) with female Bl6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J reporter mice (Jackson Laboratory; IMSR_JAX:007906), enabling Cre-dependent expression of ZsGreen1 in Kiss1 neurons.Total RNA was extracted using the Arcturus Paradise PLUS FFPE RNA Isolation Kit (Applied Biosystems, Waltham, MA, USA). Stranded total RNA sequencing libraries were constructed using the TruSeq Stranded Total RNA Library Prep Gold kit (Illumina, San Diego, CA, USA). A 1 nM pooled library (20 ul) was sequenced on an Illumina NextSeq 500 system using the NextSeq 500/550 High Output v2.5 kit (75-cycle configuration).