We collected whole heart specimens from cKO and floxed-control adult animals at 22 weeks of age to include: three male cKO, three male control, three female cKO, and three female control samples. Gene microarray analysis was conducted as previously described (34 (link)). Total RNA was isolated using TRI Reagent (Molecular Research Center, Part # TR118). After isolation, RNA was quantified and treated with RNase free DNase I (Epicentre; Part # D9905K) and repurified using RNA Clean XP magnetic beats (Beckman Coulter Part # A63987). Amplified cDNA libraries were prepared from 200 ng of DNA-free total RNA using the Universal Plus mRNA-Seq Library Prep Kit (NuGEN Technologies, Inc; Part #0509-96). Chip-based capillary electrophoresis on Agilent 2100 Bioanalyzer High Sensitivity DNA assays (Agilent Technologies; Part # 5067-4626) was used to analyze quality of the libraries, which were then quantified with the Takara Library Quantification Kit (Part # 638324).
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