Poly t oligo attached magnetic beads

Total RNA was extracted using a Spin Column Plant total RNA Purification Kit (Order No. B518661; Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. DNase digestion with Dnase I (Promega, Madison, WI, USA) was performed to remove contaminating DNA. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads (Novogene, Beijing, China) and then broken into short fragments. With these fragments as templates, cDNA were synthesized. To select cDNA fragments of 150–200 bp in length, the library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, MA, USA). Then, those fragments were selected for PCR amplification as sequencing templates. The PCR products were purified and library quality was assessed on an Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). The clustering of the index-coded samples was performed on a cBot Cluster Generation System using a TruSeq PE Cluster Kit v3-cBot-HS (Illumina, Santiago, CA, USA) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina HiSeq 4000 platform (Novogene, Beijing, China) and paired-end reads were generated. Each RNA sample was ligated with a separate adapter and sequenced together in a single run.

Total RNA was extracted from the two replicates of the drought and control plants with TRIzol Reagent (Invitrogen, 15596–026) according to the manufacturer’s instructions. The four RNA samples that were of sufficient quality were used to construct the transcriptome sequence library. The total four RNA from each sample was then pooled to one, using equivalent quantities of each sample. The transcriptome sequencing library was generated using NEBNext Ultra RNA Library Prep Kits for Illumina(NEB, USA) following manufacturer’s instructions. Following the instructions provided by Illumina, mRNA was purified from the pooled, total RNA using polyT oligo-attached magnetic beads (Novogene, China). Fragmentation buffer was added to disrupt the mRNA into short fragments. Reverse transcriptase and random primers were used to synthesise the first strand cDNA from the cleaved mRNA fragments. The second strand cDNA was synthesised using buffer, dNTPs, RNase H, and DNA polymerase I. The double strand cDNA was purified using QIAquick PCR extraction kits (QIAGEN, Hilden, Germany) and washed with EB buffer for end repair and single nucleotide A (adenine) addition. Finally, sequencing adaptors were ligated onto the fragments. The required fragments were purified by AMPure XP beads and enriched by PCR to construct a library for transcriptome sequencing.