RNA-Sequencing of nitrogen-stressed spinach tissue

Purpose: The goals of this study are to compare differentially expressed transcripts in roots and leaves of spinach plants grown under nitrogen replete and deplete conditions using transcriptome profiling (RNA-seq) Overall design: Methods: Leaf and root samples for RNA Seq analysis were collected from three independent spinach plants grown under High N (HNL1, HNL2, HNL3 for leaf samples and HNR1, HNR2, HNR3 for root samples) and Low N (HNL1, HNL2. HNL3 for leaf samples and LNR1, LNR2, LNR3 for root samples) 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. Reference genome and gene model annotation files were downloaded from SpinachBase (http://spinachbase.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 High N and Low N 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 Nitroge ntreatment in leaf tissueand root tissues was used for RNA-Seq analysis. A total of six libraries from leaf and root tissue were sequenced using the Illumina HiSeq platform. On average, 43.8 and 41.2 million raw reads were generated from leaf and root tissues in high N treatment. Across all reads for both control and drought samples, the Q20 and Q30 percentage was more than 98 and 90%, 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 85%, of which ~82 % reads uniquely mapped to the reference genome . The data generated from all libraries provided a foundation for quality analyses.