GSM7719508: Right kidney, Sham operated, 24h, rep4, RNAseq; Mus musculus; RNA-Seq
Kidney cross-section were freshly harvested at various timepoints after sham surgery or nephrectomy and tissue samples were flash frozen in liquid nitrogen. Samples were stored at -80°C until extraction. Prior to extraction the frozen kidney samples were immersed overnight at -20°C in RNAlater -ICE (Thermo Fisher AM7030) as per the manufacturer's instructions to transition the tissue to a state that was more amenable to pestle homogenisation. Total RNA was isolated from the kidney samples using the mirVana isolation kit (AM1561) as per the manufacturer's instructions except that Trizol reagent was used instead of phenol. RNA was eluted in 35µl of elution buffer. The LabChip GX Nucleic Acid Analyser (Perkin Elmer) and the Qubit RNA HS (Thermo Fisher Q32852) were used according to the manufacturer's instructions to quantify the RNA. Library preparation was performed using the Illumina TruSeq Stranded mRNA library preparation kit. Briefly, poly-A containing mRNA molecules were purified, fragmented, and converted to strand specific cDNA. This was followed by second strand cDNA synthesis, adapter ligation and PCR amplification. Resulting libraries were quality controlled using the Qubit 2.0 (Thermo Fisher Scientific) for quantitation and the LabChip GX Touch 24 (Perkin Elmer) for quality assessment. Individual libraries were combined into a final equimolar pool and then sequenced using 150bp paired end runs on the NovaSeq  system (Illumina).