GSM8421940: WT, PFC, biol rep 3; Rattus norvegicus; RNA-Seq
RNA was harvested using Trizol from tissue,1000ng of total RNA was used for the construction of sequencing libraries mRNA was purified from total RNA using poly-Toligo-attached magnetic beads. Fragmentation was carried out usingdivalent cations under elevated temperature in First Strand SynthesisReaction Buffer(5X). First strand cDNA was synthesized using randomhexamer primer and M-MuLV Reverse Transcriptase(RNase H-).Second strand cDNA synthesis was subsequently performed usingDNA Polymerase I and RNase H. Remaining overhangs were convertedinto blunt ends via exonuclease/polymerase activities. After adenylationof 3' ends of DNA fragments, Adaptor with hairpin loop structure wereligated to prepare for hybridization. In order to select cDNA fragmentsof preferentially 370~420 bp in length, the library fragments werepurified with AMPure XP system. Then PCR was performed withPhusion High-Fidelity DNA polymerase, Universal PCR primers andIndex (X) Primer. At last, PCR products were purified (AMPure XPsystem) and library quality was assessed on the Agilent Bioanalyzer2100 system