RNA-Seq of Vincetoxicum versicolor: young fresh leaves
The process of RNA library construction was as follows. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature 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 subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3 ends of DNA fragments, NEBNext adaptor with hairpin loop structure were ligated. The adaptor-ligated fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA) to select a size range of cDNA fragments of preferentially 150~200 bp. Using these purified cDNA as templates, a 150 bp paired-end library was constructed using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following manufacturers recommendations. Finally, the cDNA library was sequenced on Illumina HiSeqTM 2500 platform