Illumina HiSeq 2500 paired end sequencing; RNA seq of fluid shear stress treated mouse Pkd1-/- renal epithelial cells
RNA seq of fluid shear stress treated mouse Pkd1-/- renal epithelial cells
Pkd1-/- proximal tubular epithelial cells (PTECs) were exposed to laminar fluid shear stress (1.9 dyn/cm2) in a cone-plate device. The cone-plate device was designed for 3.5 cm cell culture dishes (Greiner Bio-One). Cells were grown on collagen-I (Advanced BioMatrix; #5005) coated dishes until confluence, followed by 24 hr serum starvation, before the start of the treatment to exclude effects of serum-derived growth-factors and to synchronize cells and cilia formation. Culture dishes were placed in the cone-plate flow system and incubated at 37°C and 5% CO2. The confluent cell monolayer of 9.6 cm2 was subjected to constant laminar (Re = 0.3) fluid shear stress for 6 hours, using 2 ml serum-free DMEM/F-12 medium containing penicillin-streptomycin, with viscosity (μ) of 0.0078 dyn s/cm2, a cone with an angle (α) of 2° and a velocity (ω) of 80 rpm, generating a fluid shear stress (τ = μω/α) of 1.9 dyn/cm2. Static control cells were incubated for the same time in equal amounts of serum-free DMEM/F12 medium containing penicillin-streptomycin at 37°C and 5% CO2. Total RNA was isolated from fluid shear stress treated Pkd1-/- PTECs or static controls (n = 4) using TRI Reagent (Sigma-Aldrich; #T9424) and purified using Nucleospin RNA Clean-up (Macherey-Nagel; #740948) according to manufacturer's protocols. Illumina mRNA-Seq Sample Prep Kit was used to process the samples according to the manufacturer's protocol. Briefly, mRNA was isolated from total RNA using the oligo-dT magnetic beads. After fragmentation of the mRNA, a cDNA synthesis was performed. This was used for ligation with the sequencing adapters and PCR amplification of the resulting product. The quality and yield after sample preparation was measured with a DNA 1000 Lab-on-a-Chip. The size of the resulting products was consistent with expected size distribution (a broad peak between 300-500 bp).
Experimental Factor: stimulus
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