GSM8098406: DSS Group Sample 3; Mus musculus; RNA-Seq
Total RNA was extracted and its quantity and quality were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Total RNA was used as input material.  mRNA was purified 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.  RNA was degraded using RNaseH.  Second strand cDNA synthesis was performed using DNA Polymerase I and dNTP.  Overhangs were converted into blunt ends via exonuclease/polymerase activities.  After adenylation of 3' ends, adaptors with hairpin loop structures were ligated.  cDNA fragments of 370~420 bp were selected using the AMPure XP system.  PCR amplification was performed, and PCR products were purified with AMPure XP beads to obtain the final library.