GSM9427911: MV411 cells, Corilagin, 48h, rep1; Homo sapiens; RNA-Seq
Total RNA was extracted by the chloroform method. Three micrograms of RNA were used as the starting material for the RNA sample preparations. The sequencing library was prepared according to the following steps: First, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Next, fragmentation was performed under elevated temperature using divalent cations in an Illumina proprietary fragmentation buffer. First strand cDNA was synthesized using random oligonucleotides and Super Script II, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Residual overhangs were converted into blunt ends via exonuclease/polymerase activity, and the enzymes were subsequently removed. After adenylation of the 3' ends of the DNA fragments, Illumina paired-end (PE) adapter oligonucleotides were ligated. To select cDNA fragments of the preferred in length, library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA). DNA fragments with adapters ligated om both ends were selectively enriched through a 15 cycle PCR reaction using the Illumina PCR Primer Cocktail. The final products were purified with the AMPure XP system and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The total concentration of the libraries was measured using the PicoGreen assay (Quantifluor-ST fluorometer, Promega, Madison, Wisconsin, USA, Quant-iT PicoGreen dsDNA Assay Kit, Invitrogen, California, USA). The effective concentration of the libraries was quantified by qPCR (StepOnePlus Real-Time PCR Systems, Thermo Scientific, Waltham, Massachusetts, USA). Multiplexed DNA libraries were normalized and pooled at equal volumes. The pooled library was serially diluted, quantified, and subsequently sequenced in PE150 mode on an Illumina sequencing platform.