GSM8013619: HN6 cells transfected with vector plasmids-1; Homo sapiens; RNA-Seq
RNA was extracted fromcells using TRIzol reagent (Invitrogen). Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA by using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature inFirst Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized usingrandom hexamer primer and M-MuLV Reverse Transcriptase, then use RNaseHtodegrade the RNA. Second strand cDNA synthesis was subsequently performed usingDNA Polymerase I and dNTP. Remaining overhangs were converted into blunt endsvia exonuclease/polymerase activities. After adenylation of 3' ends of DNAfragments, Adaptor with hairpin loop structure were ligated to prepare forhybridization. In order to select cDNA fragments of preferentially 370~420 bpinlength, the library fragments were purified with AMPure XP system(BeckmanCoulter, Beverly, USA). Then PCR amplification, the PCR product was purifiedbyAMPure XP beads, and the library was finally obtained