GSM5946859: High-fat diet PVN rep 3; Mus musculus; RNA-Seq
Control, HFD, and HFD+Mino mice (n=3/group) underwent the same diet exposure as the mice in the above groups but did not undergo metabolic testing. At the end of study, mean body mass was not different between these mice and their respective groups in the other cohorts described above. PVN tissue punches were then utilized for RNAseq. Approximately 30-60 mg of tissue sample was transferred to a safe-lock microcentrifuge tube (Eppendorf). A mass of stainless-steel beads (Next Advance, cat# SSB14B) equal to the mass of the tissue was added to the tube. Two volumes of TRI Reagent® (Zymo Research) were added to the tube and samples were immediately mixed in a bead mill homogenizer (Bullet Blender, Next Advance) for 1 min at a speed of ten. Samples were visually inspected to confirm desired homogenization and then incubated at 37°C for 5 min. The TRI Reagent® was added up to 0.6 ml, and samples were mixed in the Bullet Blender for 1 min. Total RNA was extracted using Direct-zol™ RNA Miniprep Kit (Zymo Research). Optical density values of extracted RNA were measured using NanoDrop (Thermo Scientific) to confirm an A260:A280 ratio above 1.9. RNA integration number (RIN) was measured using BioAnalyzer (Agilent Technologies) RNA 6000 Nano Kit. The cDNA libraries were prepared using the SMARTer Ultra Low Input RNA Kit for Sequencing – v4 (TAKARA Bio) and Nextera XT DNA Library Prep Kit (Illumina) as per manufacturer's instructions. The unique barcode sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. The final product was assessed for its size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies). The libraries were pooled and diluted to 3 nM using 10 mM Tris-HCl, pH 8.5 and then denatured using the Illumina protocol. The denatured libraries were loaded onto an S1 flow cell on an Illumina NovaSeq 6000 (Illumina) and run for 2X50 cycles according to the manufacturer's instructions. De-multiplexed sequencing reads were generated using Illumina bcl2fastq (released version 2.18.0.12) allowing no mismatches in the index read.