Analysis of Transcriptome Complexity via RNA-Seq in Normal and Failing Murine Hearts

RNA purification, library preparation and Illumina sequencing Left ventricular tissues were collected from male C57BL/6 mice after 1 week (hypertrophy stage, HY) and 8 weeks post trans-aortic constriction (TAC) procedure (heart failure stage, HF), respectively, and their corresponding Sham controls (Sham-HY, Sham-HF). Doppler velocity measurements of right and left carotid arteries were obtained from TACed mice in order to ensure consistent pressure-gradient generated by the procedure. A 6 to 10 fold change in velocity ratio between right and left carotid arteries after TAC was required for a successful TAC. To conduct RNA-Seq analysis, total RNAs from six TAC and Sham-operated mice at the HY stage and four TAC and corresponding Sham mice at the HF stage were obtained. The hypertrophy and heart failure status of the TAC treated animals was established based on a significant increase in heart weight and a significant reduction in ejection fractions measured by echocardiogram . Consistent with the literature , at 1-week post TAC, cardiac hypertrophy was detected while cardiac function was preserved. In contrast, at 8 weeks post-TAC, the mice demonstrated both hypertrophy and heart failure phenotypes (Online Table I). Total RNA was isolated using the mirVana kit (Applied Biosystems), according to the manufacturer’s instructions. We used the standard Illumina protocol to prepare libraries for RNA-Seq (http://www.illumina.com/support/documentation.ilmn). Briefly, 10μg total RNA was first processed via poly-A selection and fragmentation. We generated first-strand cDNA using random hexamer-primed reverse transcription and subsequently used it to generate second-strand cDNA using RNase H and DNA polymerase. Sequencing adapters were ligated using the Illumina Paired-End sample prep kit. Fragments of ~200 bp were isolated by gel electrophoresis, amplified by 15 cycles of PCR and sequenced on the Illumina Genome Analyzer II in the paired-end sequencing mode (2x72 bp or 2x76 bp reads).