OVXTA rep3
In the library preparation process, RNA integrity is first assessed using the RNA Nano 6000 Assay Kit of the Agilent 2100 Bioanalyzer system. mRNA is then purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation is performed using divalent cations under elevated temperature. First strand cDNA is synthesized using random hexamer primers and M-MuLV Reverse Transcriptase, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. The remaining overhangs are converted into blunt ends, and adenine is added to the 3' ends of DNA fragments before ligation of adaptors with hairpin loop structures. The cDNA fragments are purified using the AMPure XP system to select fragments of 370~420 bp in length. PCR is conducted using Phusion High-Fidelity DNA polymerase, and the PCR products are purified again with the AMPure XP system. The library quality is assessed using the Agilent 2105 Bioanalyzer system.