GSM9097184: PC3 cells, siCtrl4; Homo sapiens; RNA-Seq
Total RNA was isolated from samples using TRIzol reagent. Atotal amount of 1 µg RNA per sample was used as input material for the RNA  sample preparations. Sequencing libraries were generated using NEBNext® UltraTM  RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's  recommendations and index codes were added to attribute sequences to each sample.  Briefly, mRNA was purified from total RNA using poly-T oligo-attached  magnetic beads. Fragmentation was carried out using divalent cations under elevated  temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand  cDNA was synthesized using random hexamer primer and M-MuLV Reverse  Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently  performed using DNA Polymerase I and RNase H. Remaining overhangs were  converted into blunt ends via exonuclease/polymerase activities. After adenylation of  3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were  ligated to prepare for hybridization. In order to select cDNA fragments of  preferentially 250~300 bp in length, the library fragments were purified with AMPure  XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA)  was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5  min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA  polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products  were purified (AMPure XP system) and library quality was assessed on the Agilent  Bioanalyzer 2100 system.