RNA-Sequencing of salt-stressed watermelon seedlings

Purpose: The goals of this study are to compare differentially expressed transcripts in seedlings of watermelon during salt stress using transcriptome profiling (RNA-seq) Overall design: Methods: Leaf samples for RNA Seq analysis were collected from three independent plants at 7 hours after initiation of salt stress from control (CT2_1, CT2_2, CT2_3) and salt-stressed plants (TT2_1, TT2_2, TT2_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.