RNA-Seq of Scophthalmus maximus: liver
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 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, Adaptor with hairpin loop structure were  ligated to prepare for hybridization. In order to select cDNA fragments  of preferentially 370~420 bp in length, the library fragments were  purified with AMPure XP system. 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.