GSM9305533: Zeb1_200H_pituitary_2_youngCohort; Mus musculus; RNA-Seq
Total RNA was extracted from mouse tissues using TRI Reagent (ThermoFisher) according to the manufacturer's protocol with the following modifications. Mouse tissues were rapidly dissected after CO2-based euthanasia and flash frozen in Eppendorf tubes in liquid N2. Tissue was transferred to a 15 mL conical tube, 1–2 mL of TRI Reagent was added, and the tissue was homogenized with a TissueRuptor (Qiagen) and disposable probes. Samples were transferred to Eppendorf tubes (1 mL per tube) and phase separated with 200 μL chloroform (J.T. Baker Analytical). After isopropanol precipitation and two 70% ethanol washes, total RNA was resuspended in water. Prior to library preparation, ribosomal RNA depletion was performed using QIAseq FastSelect -rRNA HMR KIT oligos (Qiagen) by mixing 14 μL of RNA input (containing 0.5 μg of hypothalamus, pituitary, or ovary total RNA), 1 μL FastSelect reagent (Qiagen), and 10 μL Frag & Prime Buffer (Watchmaker Genomics) and incubating under the following conditions: 85°C 10 minutes, 75°C 2 minutes, 70°C 2 minutes, 65°C 2 minutes, 60°C 2 minutes, 55°C 2 minutes, 37°C 2 minutes, 25°C 2 minutes, 4°C. Samples were then carried forward to the First Strand Synthesis step of the Watchmaker RNA Library Prep Kit (Watchmaker Genomics). Libraries were sequenced on the Illumina NovaSeq platform with 50 nt paired-end reads.