*4.2. NGST Sequencing*

Total RNA was extracted using a Plant RNA Kit (OMEGA Bio-Tek, Norcross, GA, USA). RNA concentration was measured using NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA).mRNA was purified from total RNA with poly-T oligo-attached magnetic beads. Samples underwent an RNA-seq analysis involving three biological replicates per sample. The fragmentation step was completed with divalent cations in the NEBNext First Strand Synthesis Reaction Buffer (5×) at an elevated temperature. First-strand cDNA was synthesized with a series of random hexamer primers and reverse transcriptase, and second-strand cDNA was subsequently produced with DNA Polymerase I and RNase H. The cDNA libraries were constructed by ligating cDNA fragments to sequencing adapters and amplifying fragments by PCR. The libraries were then sequenced with the Illumina HiSeq 2000 platform (Nanxin Bioinformatics Technology Co., Ltd., Guangzhou, China).
