RNA-Seq of Sillago sihama: hypoxia  1h-treated fish gill bioreplicate 2
Fragmentation was carried out using divalent cations under elevated  temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First  strand cDNA was synthesized using random hexamer primer and M-MuLV Leading  Edge Genomic Services & Solutions 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,  NEBNext Adaptor with hairpin loop structure were ligated to prepare for  hybridization. In order to select cDNA fragments of preferentially 250~300 bp  in length, the library fragments were purified with AMPure XP system