R200H-2 (CRX870196)
RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system. 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, then use RNaseH to degrade the RNA.Second strand cDNA  synthesis was subsequently performed using DNA Polymerase I and dNTP. 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 (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  XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.