Transcriptome analysis of flag leaf during its senescence in wheat
The flag leaf was collected at the heading stage, first flowering date (0DAA), 15 days after anthesis (DAA), 25DAA, and 30DAA. At each time point, tissues were harvested from fifteen randomly selected tillers. Total RNA was extracted using the RNA extraction kit, following the manufacturer’s protocol (Bioteke, Beijing, China). Ten equimolar high-quality RNA samples for each time point were mixed. The library was constructed using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s protocol. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads, and then fragmented using divalent cations under elevated temperature . 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 to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 ? for 15 min followed by 5 min at 95 ? 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.