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.

为分析Gq受体（Gq-receptor）介导的肥厚型转录谱，我们对10~11周龄的FVB/NJ小鼠采用植入式微渗透泵（micro-osmotic pump，Alzet，型号1002）分别以30 mg/kg/天的剂量给予苯肾上腺素（phenylephrine）、0.5 mg/kg/天的剂量给予血管紧张素II（angiotensin II），持续给药72小时。采用RNeasy纤维组织迷你试剂盒（Qiagen）并严格遵循制造商说明书，从分离得到的心肌细胞中提取总RNA（total RNA）。以总RNA为起始原料，通过寡聚dT（oligo-dT）纯化去除核糖体RNA（rRNA）以富集mRNA，随后依次进行随机引物反转录、第二链cDNA合成、连接用于Illumina测序的索引接头，最终由所得双链DNA构建测序文库。由明尼苏达大学基因组核心实验室使用Illumina HiSeq 2500平台完成125 bp双端链特异性RNA测序，每个样本可获得1000万至2000万条reads。