De novo transcriptome assembly of common wild rice (Oryza rufipogon Griff.) and discovery of drought-response genes in root tissue based on transcriptomic data
In this study, the shoot (CL) and root (CR) tissues of control plants and root tissue of the treated group (15% PEG-6000 stress for 24 h) (DR) were harvested for RNA extraction. RNA samples were extracted from five plants, and each sample was analyzed twice. Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions, and the RNA quality was verified using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The RNA Integrity Number (RIN) values for each of the samples were all greater than 8. For transcriptome sequencing libraries construction, NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) was used. First, mRNA was purified from the pooled total RNA using polyT oligo-attached magnetic beads and fragmented using an RNA fragmentation buffer. First-strand cDNA was synthesized using buffer, dNTPs, random hexamer primers and reverse transcriptase, and the second cDNA strand was synthesized using buffer, dNTPs, RNase H, and DNA polymerase I. Then the double-stranded cDNA was purified using the GeneJET PCR purification kit, then washed with EB buffer and submitted to end repair and single nucleotide adenine addition. Finally, adaptors were added to the fragments, and then were purified with AMPure XP beads and enriched by PCR amplification. After cluster generation, the sequencing libraries were sequenced by Illumina HiSeq 2000 platform.