GSM4403082: Control aorta rat 46; Rattus norvegicus; RNA-Seq
The rat aortas were dissected free from the level of the renal arteries and up to the heart, connective tissue and blood were removed by gentle manipulation and a rinse with sterile saline. The aorta was instantly snap frozen in liquid nitrogen. Total RNA from the aorta was extracted with TRIzol (Sigma-Aldrich, St. Louis, MO). Purified total RNA was treated with the Qiagen RNase-free DNase set followed by purification on miRNeasy columns (Qiagen, Germantown, MD). Two rounds of enrichment of polyadenylated RNA were then performed using the Poly(A)Purist MAG kit (Ambion, Fisher Scientific, MA) followed by RNA purification on RNAClean XP beads (Agencourt, Beckman Coulter). The RNA sample was fragmented on ice by adding fragmentation buffer to a final concentration of 50 mM Tris·HCl (pH 8) and 5 mM MgCl2 followed by incubation at 95°C for 3 min and 20 s. The sample was then immediately placed back on ice, and EDTA was added at 10 nM to stabilize RNA. Fragmented RNA was purified on RNAClean XP beads (Agencourt) and eluted in 13 ul diethylpyrocarbonate-treated H2O. cDNA synthesis and ligation of an adapter to the 3=-end of the cDNA was performed (21). The first-strand synthesis and adapter ligation steps result in a cDNA pool containing adapters both at the 5=- and 3=-ends, which can be used for subsequent PCR and Illumina sequencing. Three microliters of PCR_forward (10 uM) primer (5=-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCT ACACGACGCT-3=), 10 ul Phusion 5 x HF buffer, 1 ul of 10 mM dNTPs, 26.5 ul H2O, 1 ul Phusion DNA polymerase, 2.5 ul indexing primer (10 uM, 5=-CAAGCAGAAGACGGCATACGAGATNNNNN- NGTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCT-3=; with NNNNNN indicating the index sequence), and 6 ul purified linker ligated cDNA were mixed and run with the following PCR program: 98°C for 3 min, (98°C for 80 s, 64°C for 15 s, and 72°C for 30 s) x 4, (98°C for 80 s and 72°C for 45 s) x 10, 72°C for 5 min, and hold at 4°C. The resulting sequencing libraries were quantified on a Bioanalyzer 2100 (Agilent Technologies) using a DNA1000 chip (Agilent) for smear analysis and molarity calculation. Samples were mixed in equimolar amounts, and PCR amplicons in the 200- to 600-bp size range were extracted from a 2% E-Gel SizeSelect Agarose Gel in an E-Gel Electrophoresis System (Life Technologies, Paisley, UK). The eluate was purified using Agencourt Ampure XP beads (Beckman Coulter). The final sample was again run on a DNA1000 chip (Agilent) in a Bioanalyzer 2100 for smear analysis.
GEO
GSM4403082