GSM9468069: p_941_T; Mus musculus; RNA-Seq
Total RNA was harvested using RNeasy extraction kit (Qiagen 74104) 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 treatment. After adenylation of the 3' ends of DNA fragments, Adaptor with hairpin loop structure was ligated to prepare for hybridization. In order to select cDNA fragments in the reange of 370-420 bp, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, using the Universal and Index (X) primers. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.