GSM2877962: EV_2; Homo sapiens; RNA-Seq
Total RNA was extracted using TRIzol reagent (15596-018, Ambion life technologies) according to the manufactures protocol.  2,5 mL of TRIzol reagent was used . The cells were lysed and homogenized in 15 mL tube(s) (Falcon). 0.2 vol. of chloroform (Chloroform stab./Amylene, Biosolve) was added to the homogenate and the tube(s) were shaken vigorously for 20 seconds. The tube(s)  were incubated for 2-3 minutes at room temperature and centrifuged  (Hettich, rotanta 46 RS) for 1 hour at 4 C . Approximately 70% of the upper aqueous phase was transferred to a clean 15 mL tube and 0.5x volume of isopropanol  (33539, Sigma-Aldrich, ) was added. The tube(s) were incubated overnight at -20 C and centrifuged for 30 minutes at 4 C. The supernatant was removed and the pellet was washed twice with 80% ethanol  (32221-2.5L, Sigma-Aldrich). The total RNA pellet was air-dried for 10 minutes and dissolved  in nuclease free water (AM9937, Ambion life technologies). Quality and quantity of the total RNA was assessed by the 2100 Bioanalyzer (Agilent) using a Nano chip (Agilent). Sequencing libraries were generated using the TruSeq RNA Sample Preparation v2 Guide (Illumina Part # 15026495 Rev. F) according to the manufacturer's instructions. Briefly, polyadenylated RNA from intact total RNA was purified using oligo-dT beads. Following purification the RNA was fragmented, random primed and reverse transcribed using SuperScript II Reverse Transcriptase (Invitrogen, part # 18064-014). Second strand synthesis was performed using Polymerase I and RNaseH. The resulting cDNA was converted to blunt end fragments using an end repair mix (Illumina), followed by 3' end adenylation and ligated to Illumina Paired-end sequencing adapters and subsequently amplified by 16 cycles of PCR. The libraries were analyzed on a 2100 Bioanalyzer (Agilent) using a 7500 chip(Agilent) and diluted to 10nM.