GSM2333488: U87 brain invasive clone 2; Homo sapiens; RNA-Seq
Briefly, 0.2x volumes of chloroform (Chloroform stab./Amylene, Biosolve) was added to the Trizol homogenate and the tube(s) (Falcon, 15mL) were shaken vigorously. 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 8 minutes and dissolved in an appropriate volume of nuclease-free water (AM9937, Ambion Life Technologies) and quantified using NanoDrop UV-VIS Spectrophotometer. The total RNA was further purified using the MinElute Cleanup Kit (74204, Qiagen) according to the manufacturer's instructions. Quality and quantity of the total RNA were assessed by the 2100 Bioanalyzer using a Nano chip (Agilent, Santa Clara, CA). Total RNA samples having RIN>8 were subjected to library generation. Strand-specific libraries were generated using the TruSeq Stranded mRNA sample preparation kit (Illumina Inc., San Diego, RS-122-2101/2) according to the manufacturer's instructions (Illumina, Part # 15031047 Rev. E). 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) with the addition of Actinomycin D. Second strand synthesis was performed using Polymerase I and RNaseH with replacement of dTTP for dUTP. The generated cDNA fragments were 3' end adenylated and ligated to Illumina Paired-end sequencing adapters and subsequently amplified by 12 cycles of PCR. The libraries were analyzed on a 2100 Bioanalyzer using a 7500 chip (Agilent, Santa Clara, CA), diluted and pooled equimolar into a 10nM multiplex sequencing pool and stored at -20 °C. The libraries were sequenced with 65 base single reads on a HiSeq 2500 using V4 chemistry (Illumina Inc., San Diego).