in vivo 3-day RNA Seq: vehicle, angiotensin II, and phenylephrine
To analyze Gq-receptor hypertrophic transcriptional profiles, 10 to 11 week old FVB/NJ mice were dosed at 30 mg/kg/day for phenylephrine and 0.5 mg/kg/day for angiotensin II using implanted micro-osmotic pumps (Alzet, model 1002) for 72 hours. Total RNA was harvested from isolated myocytes using RNeasy Fibrous Tissue Mini Kit (Qiagen), following manufacturer’s instructions. Using total RNA as input, rRNA was removed via oligo-dT purification to enrich for mRNA, followed by random-primed reverse-transcription, second-strand cDNA synthesis, ligation of indexed adaptors for Illumina sequencing, and subsequent library creation from the resulting double-stranded DNA. 125 base pair paired end stranded RNA Sequencing was performed by the University of Minnesota Genomics Core using the Illumina HiSeq 2500 resulting in 10-20 million reads per sample.