SRA

Purpose: The goals of this study are to compare differentially expressed transcripts in leaves of watermelon during drought stress using transcriptome profiling (RNA-seq) Overall design: Methods: Leaf samples for RNA Seq analysis were collected from three independent plants at 8 days after initiation of drought stress from control (CT1_1, CT1_2, CT1_3) and drought-stressed plants (DT_1, DT_2, DT_3) were flash-frozen in liquid nitrogen for further analysis. Results: The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, the libraries were sequenced on an Illumina Hiseq platform, and 150 bp paired-end reads were generated. Raw reads of fastq format were processed to obtain clean reads by removing the adapter, reads containing ploy-N, and low quality reads from raw data. At the same time, Q20, Q30, and GC content, the clean data were calculated. Watermelon reference genome (cultivar Charleston Gray) and gene model annotation files were downloaded from CuGenDB (http://cucurbitgenomics.org/). Index of the reference genome was built using Bowtie v2.2.3, and paired-end clean reads were aligned to the reference genome using TopHat v2.0.12. : HTSeq v0.6.1 was used to count the reads mapped to each gene. Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated from mapped read by featureCounts. Differential expression analysis of control and drought-stressed conditions (three biological replicates per tissue per treatment) was performed using the DESeq R package (1.18.0) (Anders and Huber, 2010). Genes with P-value < 0.05 found by DESeq were assigned as differentially expressed. Conclusions: The transcriptomic changes induced by drought stress in leaf tissue at 8DAT was used for RNA-Seq analysis. A total of six libraries from leaf tissue (CT-1, CT-2, CT-3 for control and DT-1, DT-2, DT-3 for drought stress) were sequenced using the Illumina HiSeq platform. On average, 45.53 to 43.47 million raw reads were generated from leaf tissues in both treatments. Across all reads for both control and drought samples, the Q20 and Q30 percentage was more than 98 and 94%, respectively (sequencing error rate was less than 0.02%), and GC content for the libraries was ~45%. Among all the libraries, the ratio of total mapped reads was above 97%, of which ~92 % reads uniquely mapped to the reference watermelon genome for control and drought-stressed samples. The data generated from all libraries provided a foundation for quality analyses. The RNA-Seq analysis identified 3971 differentially expressed (p <0.05) genes in the leaf tissue of drought-stressed plants, of which a total of 1513 genes were upregulated and 2458 genes were downregulated.