GSM8244149: ENL_Y78A; Homo sapiens; RNA-Seq
Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). 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 ReverseTranscriptase(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.