Total RNA from three different tissues, including leaves, stems, and roots, was extracted from the two biological replicates of the drought-treated and control plants with TRIzol Reagent (Invitrogen, 15596-026) according to the manufacturer's instructions. Each DGE library was constructed by pooling RNA from four plants. We used 3 μg of total RNA per sample as input material for RNA library preparation. The DGE sequencing libraries were generated using NEBNext Ultra RNA Library Prep Kits for Illumina (NEB, USA). Following the instructions provided by Illumina, mRNA was purified from the pooled, total RNA using oligo dT magnetic beads (Novogene, China). Fragmentation buffer was added to reduce the mRNA molecules to short fragments. Reverse transcriptase and random primers were used to synthesize the first-strand cDNA from the mRNA fragments. Second-strand cDNA was synthesised using buffer, dNTPs, RNase H, and DNA polymerase I. The double-stranded cDNA was purified using QIAquick PCR extraction kits (QIAGEN, Hilden, Germany) and washed with EB buffer for end repair and addition of a terminal A (adenine) nucleotide. Illumina sequencing adapters were then ligated onto the cDNA fragments. The cDNA fragments containing terminal adapters were purified by AMPure XP beads and enriched by PCR to construct the library for RNA sequencing.
GEO Accession
GSM3885409