GSM5949121: M019R TS IFNg_24h; Homo sapiens; RNA-Seq
Total RNA was extracted using TRIzol reagent (15596-018, Ambion life technologies)  according to the manufactures protocol. 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  (4500 RCF) (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 (4500 RCF)  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  RNeasy Mini kit (74106, Qiagen), including an on-column DNase digestion (79254, Qiagen),  according to the manufacture's instructions. Quality and quantity of the total RNA was 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 multiplex sequencing pools. The  libraries were sequenced with 65 base single reads on a HiSeq2500 using V4 chemistry (Illumina Inc., San Diego). TruSeq Stranded mRNA sample preparation kit