design: In this project, total RNA of each sample was extracted from the barley leaves according to the instruction manual of the TRlzol Reagent (Life technologies, California, USA). RNA integrity and concentration were checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). The mRNA was isolated by NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490). The cDNA library was constructed following the manufacturers instructions of NEBNext Ultra RNA Library Prep Kit for IlluminaNEB, E7530and NEBNext Multiplex Oligos for IlluminaNEB, E7500). In briefly, the enriched mRNA was fragmented into approximately 200nt RNA inserts, which were used to synthesize the first-strand cDNA and the second cDNA. The double-stranded cDNA were performed end-repair/dA-tail and adaptor ligation. The suitable fragments were isolated by Agencourt AMPure XP beads (Beckman Coulter, Inc.), and enriched by PCR amplification. Finally, the constructed cDNA libraries of the barley were sequenced on a flow cell using an Illumina HiSeq sequencing platform.