RNA-seq of primary rat hypothalamic neurons by short- and long-term estrogen stimulation and withdrawal
RNA-seq of primary rat hypothalamic neurons by short- and long-term estrogen stimulation and withdrawal
Primary hypothalamic neurons were prepared from embryonic day 18 (E18) Sprague-Dawley rats. Briefly, hypothalamus was rapidly dissected under sterile conditions, minced into small fragments, and digested using Pierce Primary Neuron Isolation Kit. The digestion was terminated by adding an equal volume of B-27 Neurobasel Plus Medium. Cells were gently triturated using a fire-polished Pasteur pipette, passed through a 70-µm cell strainer, and plated onto 100 µg/mL poly-D-lysine-coated culture dishes at a density of 1 × 105 cells/cm². After 4 h, the medium was replaced. Cultures were maintained at 37 °C in a humidified incubator with 5% CO₂, and half of the medium was replaced every 3 days. To keep the Kiss1 gene activity, 0.1 nM E2 was always added in medium. 1 nM (final concentration, similarly hereinafter) E2 for 24 h or 60 h to model the short and long term estrogen exposure, then for additional 48 h culturing after E2 withdrawal. Total RNA of 5 x 107 cells were extracted using TRIZOL (Thermo Fisher Scientific). Nanodrop 2000 (Thermo Fisher Scientific) and Agilent Bioanalyzer 2100 (Agilent) were used to evaluate RNA concentration and quality. 5 μg of RNA from different experimental groups was prepared cDNA library using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB) following the manufacturer's instructions.