RNA-Seq of leaf: tobacco seedling
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 Buffer5X. Next Adaptor with hairpin loop structure were ligated to prepare for hybridization. Then 3 l USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA  before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.