73397-E-2 (CRX2174742)
Total RNA was extracted from callus tissues and qualified through rigorous quality control. The poly(A)+ mRNA was enriched from total RNA using Oligo(dT) magnetic beads. The enriched mRNA was fragmented into approximately 300 bp pieces using divalent cations. First- and second-strand cDNA were synthesized using the fragmented mRNA as template. The double-stranded cDNA was then end-repaired, 'A'-tailed, and ligated with sequencing adapters. The library fragments were purified and size-selected using AMPure XP beads, followed by PCR amplification. The final library was quantified using Qubit 2.0, and the insert size distribution was assessed with the Agilent 5400 Fragment Analyzer system. The effective library concentration was accurately quantified by qPCR (>2 nM) to ensure quality for sequencing.