GSM9191164: Ctrl_3.; Homo sapiens; RNA-Seq
Total RNA was extracted from Human basal cells using Trizol (15596018CN, Invitrogen), and RNA extraction was performed according to the manufacturer's instructions. Subsequently, the extracted RNA was treated with Dnase I (Invitrogen, Life Technologies, USA) to remove any contaminating DNA. RNA integrity was assessed using theRNA Nano 6000 Assay Kit of the Bioanalyzer2100 system (Agilent Technologies, CA, USA). Total RNA was used as input material for the RNA sample preparations. 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 First StrandSynthesisReaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLVReverse Transcriptase(RNaseH-). Second strand cDNA synthesis was subsequently performed usingDNAPolymeraseI and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3'ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNAfragments of preferentially370~420 bp in length, the library fragments were purified with AMPure XP system(Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XPsystem) and library quality was assessed on the Agilent Bioanalyzer2100 system.