GSM6591455: L_PS8; Rattus norvegicus; RNA-Seq
mRNA was purified from total RNA using polyT, oligo-attached magnetic beads, followed by fragmentation using divalent cations in NEBNext 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 then performed using DNA polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of 250~300 bp, the library fragments were purified with AMPure XP system (Beckman Coulter). Then, 3 μl USER Enzyme (New England BioLabs) was used with size-selected, adaptor-ligated cDNA at 37° C for 15 min, which was followed by another incubation of 5 min at 95 °C before PCR. PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Lastly, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina platform