title: Illumina HiSeq 2500 sequencing; Left ventricle transcriptomic analysis reveals the initial decline in V̇O2peak from its lifetime  apex associates with connective tissue accumulation
design: Left ventricle transcriptomic analysis reveals the initial decline in V̇O2peak from its lifetime  apex associates with connective tissue accumulation
library_construction_protocol: V̇O2peak testing To teach the rat how to properly maintain running at the front of the treadmill, all rats began acclimatization to a belt-driven treadmill at 7 wks of age. During acclimatization, rats walked at 15 m/min for five minutes, 3x per week for 2 weeks, until 10 wks of age when the first V̇O2peak test was performed. Importantly, all V̇O2peak testing was performed by a blinded individual using a Metabolic Modular Treadmill (Columbus Instruments, Columbus, OH). At test completion, RER values ranged from 0.90 to greater than 1.00, thus a true V̇O2max was not measured in every test and values are reported as V̇O2peak. No rats reached a plateau in V̇O2, possibly due to our testing procedures which progressively increased speed rather than grade. While we measured V̇O2peak rather than V̇O2max, it is documented that within the same untrained animal, results from maximal oxygen tests whereby a plateau is not observed agree closely with those in which a true max or plateau in oxygen consumption is achieved. At the time of sacrifice, the left ventricle was removed, rapidly frozen in liquid nitrogen, and stored at-80°C until processing. Tissue was homogenized in Trizol using stainless steel beads and Tissuelyser LT. RNA was subsequently extracted using the Trizol method and Qiagen RNeasy kit according to the manufacturer’s instructions, and the resultant concentration was determined using Nanodrop 1000. RNA was eluted in 40 μL of RNase-free water. RNA integrity was confirmed using the BioAnalyzer 2100 automated electrophoresis system prior to cDNA library construction. RNA-seq preparation, including cDNA library construction, was carried out at the University of Missouri DNA Core following manufacturer's protocol using the Illumina TruSeq RNA sample preparation kit v2. Poly-A containing mRNA was isolated from 2 μg of total RNA, RNA fragmentation was carried out, and double-stranded cDNA was produced from fragmented RNA. Lastly, identifier adaptors were ligated to the ends for identification purposes. The final construct of each purified library was evaluated with the BioAnalyzer 2100 automated electrophoresis system, quantified, and diluted in accordance with Illumina's protocol for HiSeq 2000
attributes.tag: Experimental Factor: 17
attributes.value: age
attributes.tag: Experimental Factor: no exercise wheel access
attributes.value: exercise test