photosynthetically active leaf tissue_biological replicate 2
A total amount of 1 g RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext UltraTM RNALibrary Prep Kit for Illumina (NEB, USA) following manufacturers recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA waspurified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First StrandSynthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesiswas subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of3 ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp inlength, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 l USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 C for 15 min followed by 5 min at 95 C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers andIndex (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system