alias: GSM2051539
title: GSM2051539: obese_2; Ovis aries; RNA-Seq
library_construction_protocol: Harvesting of heart tissue was conducted during the second breeding season, when the selected animals were 21 months old. All animals were given two 20 mg injections of Prostaglandin F2α (PGF2α, 5 mg/mL Lutalyse; Pfizer Animal Health, NY, USA), 11 days apart, to induce luteolysis and synchronize the initiation of the follicular phase in cycling females. Ewes were euthanized after the second PGF2α injection, during the presumptive follicular phase, by administration of a barbiturate overdose (Fatal Plus, Vortech Pharmaceuticals, Dearborn, MI).  Hearts were dissected out and weighed, then myocardium was harvested from a randomly selected subset of four animals per group (n=4 for each of the control, BPA, obese, and ObBPA groups) and quick frozen until processed. Total RNA was extracted from the frozen myocardium using TRIzol Reagent (Life Technologies) according to the manufacturer’s instructions. The concentration of RNA from each sample was measured by NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Scientific). OD 260/280 values were 1.87 - 1.96 for the RNA samples. The integrity of each RNA sample was monitored through Agilent BioA RNA Integrity Number (RIN). RNA-Seq was performed by the Genomic Sequencing Core (GSC) in the University of Cincinnati. Using the TruSeq RNA sample preparation kit (Illumina), one µg of total RNA with RIN (Agilent 2100 Bioanalyzer) ³ 7.0 was used to purify poly-A containing mRNA using poly-T oligo-attached magnetic beads. The purified mRNA was enzymatically fragmented and random hexamers-primed for the first and second strand cDNA synthesis, followed by purification using Agencourt AMPure XP beads (Beckman Coulter).The double-strand cDNA with overhangs was converted into blunt ends in the end repair procedure, and adenylated to add a single "A" nucleotide at 3' ends to prevent them from self-ligation in the following ligation step. The AMPure XP beads-purified fragments were then ligated to sample-specific indexing adapters, and enriched by 10 cycles' of PCR using adapter-specific primers.One µl of purified PCR product (sequencing library, total 30 µl) was analyzed by Bioanalyzer (Agilent) using the DNA 1000 chip to check the DNA fragment size (~260 bp) and yield. To accurately quantify the library concentration for the clustering, the library was 1:104-106 diluted in dilution buffer (10 mM Tris-HCl, pH 8.0 with 0.05% Tween 20), and qPCR analyzed by the Kapa Library Quantification kit (Kapabiosystem) using ABI's 9700HT real-time PCR system
identifiers.namespace: GEO
identifiers.id: GSM2051539