GSM7093507: paVICs, dantrolene+ethanol, rep1; Sus scrofa; RNA-Seq
RNA was harvested into TRIzol and purfied using a Direct-zol RNA microprep spin kit with DNAse treatment. RNA was eluted into RNAse free water. 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 Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strandsynthesis was performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. 3' ends of fragments were adenylated and Adaptor with hairpin loop structure were ligated. Library fragments (~370-420 bp) were purified with AMPure XP system (Beckman Coulter, Beverly, USA). PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCRprimers and Index (X) Primer. PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.