title: RNAseq_shoot
design: Sequencing libraries were generated using the NEBNext Ultra RNA Library Prep Kit for Illumina following the manufacturer's protocol, and index codes were assigned to each sample. mRNA was isolated from total RNA with poly-T magnetic beads and fragmented using divalent cations at high temperatures. First-strand cDNA synthesis was conducted using random primers and M-MuLV Reverse Transcriptase, followed by second-strand synthesis with DNA Polymerase I and RNase H. Overhangs were blunted, and 3' ends were adenylated. NEB Next Adaptors with hairpin loop structures were ligated to the cDNA. The library fragments were then size-selected to 370-420 bp using the AMPure XP system. USER Enzyme was applied to adaptor-ligated cDNA, followed by PCR with Phusion High-Fidelity DNA polymerase and primers. PCR products were purified and the library quality was assessed on the Agilent 5400 system and quantified by QPCR. The qualified libraries were pooled and sequenced on an Illumina HiSeq 2000 platform using a PE150 approach by Novogene Bioinformatics Technology Co., Ltd. Each sample undergoes independent library construction with three biological replicates.