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Drought stress is the main abiotic factor affecting rice production. Rain-fed upland rice which is grown on unbounded fields and totally dependent on rainfall for moisture is more prone to drought stress compared to rice from other ecosystems. However, upland rice has adapted to this limited water condition, thus are more drought tolerant than rice from other ecosystems. We performed the first transcriptome sequencing of drought tolerant indica upland rice cultivar Kuku Belang to identify differentially expressed genes related to drought tolerance mechanism. Raw reads for non-treated and PEG-treated Oryza sativa subspecies indica cv. Kuku Belang were deposited in the NCBI SRA database with accession number SRP074520 (https://www.ncbi.nlm.nih.gov/sra?term=SRP074520).
Value of data
Transcriptome data of Oryza sativa subspecies indica cv. Kuku Belang were generated from the polyA-enriched cDNA libraries prepared from total RNA extracted from two weeks old seedlings treated with PEG (treated sample) and distilled water (non-treated sample). Short reads were filtered, processed, assembled and analysed as describe in the next section. Raw data for this project were deposited in the NCBI SRA database with accession number SRP074520 (https://www.ncbi.nlm.nih.gov/sra?term=SRP074520).
Seeds of O. sativa indica cv. Kuku Belang obtained from Malaysian Agricultural Research and Development Institute (MARDI), Seberang Prai were sterilised, germinated, and sown in glass house (2°55′14.5′′N 101°47′01.4′′E) with the temperature at 26/22 °C (day/night), 75/70% humidity, day length of 12 h, and light intensity of 700 µmol m−2 s−2. To mimic drought stress, two weeks old seedlings were treated with PEG by immersing its roots for 6,12, 18, 48, 72, and 96 h in 20% PEG-6000 solution whereas for non-treated samples, the roots were immersed in distilled water. Samples were collected at the designated time points and frozen in liquid nitrogen before being stored at −80 °C.
Exact masses of total RNA extracted from rice seedlings treated with 20% PEG-6000 for 6,12,18,48,72 and 96 h were combined into one sample (treated sample). Similarly, exact masses of total RNA extracted from rice seedlings treated with distilled water for 6,12,18,48,72 and 96 h were combined into one sample (non-treated sample). Total RNA was extracted using TRIzol reagent as described by the manufacturer (Life Technologies). Total RNA purity was confirmed using Nanodrop 1000 (Thermo Fisher Scientific Inc., USA) whereas total RNA integrity was confirmed using 1% agarose gel electrophoresis. DNA contamination was removed using RNAse-free DNase kit as described by the manufacturer (Thermo Scientific). Both of treated and non-treated samples were sent for sequencing at Malaysian Genome Institute (MGI).
PolyA-enriched cDNA library was prepared using TruSeq Stranded Total RNA Sample Preparation with Ribo-Zero Plant kit as described by the manufacturer (Illumina). PEG-treated sample was indexed using TruSeq Adapter Index 14 whereas non-treated sample was indexed using TruSeq Adapter Index 7. Quality of cDNA library prepared were analysed using Agilent Technologies 2100 Bioanalyzer (Agilent Technologies, USA). Clustering was performed using cBot (version 1.4) and TruSeq PE Cluster v3 kit (Illumina). Paired-end sequencing of 101 bp was then performed using Illumina HiSeq™ 2500 and TruSeq SBS v3 kit (Illumina).
High quality raw reads with Phred score ≥ 30 generated from sequencing of PEG-treated and non-treated samples were kept for assembly. Genome-guided assembly was performed using the Tuxedo  protocol whereby the high quality raw reads of both samples were mapped independently to the reference genome used which is the O. sativa subspecies indica genome ASM465v1.15 using TopHat (v2.0.4) . The alignment files of both samples were then fed independently to Cufflink (v2.0.1) . Next, the assembled transcripts from both samples were merged to produce final transcriptome assembly using Cuffmerge . Cuffmerge  was also used to merge the final transcriptome assembly with the reference genome annotation. CuffDiff was used to quantify transcripts abundance (FPKM) in both samples and identify differentially expressed genes according to gene expression level and statistical significance test. Genes with log2 fold change ≥ 2, p-value ≤ 0.001 and q-value ≤ 0.05 were considered differentially expressed. Expression plots such as scatter plot (Fig. 1) and density plot (Fig. 2) were generated using CummeRbund (v2.0.0) . Heatmap was generated using Cluster 3.0  and Treeview (v1.1.6r4)  (Fig. 3). Table 1 shows the sequencing and RNA-seq statistics. Lists of differentially expressed genes were provided as Supplementary material.
This research is funded by grant PJ008574 from National Academy of Agricultural Science, RDA, Suwon, Republic of Korea.