Computational protocol: RNA-seq analysis reveals the role of red light in resistance against Pseudomonas syringae pv. tomato DC3000 in tomato plants

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Protocol publication

[…] The raw reads generated from the sequencing machines were cleaned by discarding the adaptor sequences and low-quality reads and filtering the reads with an unknown nucleotide percentage greater than 5%. The mapping of clean reads (from the single-end RNA-seq library) onto the tomato genome (The International Tomato Annotation Group Solanum lycopersicum protein reference version 2.0 reference) was conducted with Bowtie using the default parameters. The sequences from the Illumina sequencing were deposited in the NCBI Sequence Read Archive database (Accession GSE64087, Gene expression was quantified as the total number of reads for each sample that uniquely aligned to the reference. The functional annotation software Blast2go Program [] was used to assign gene ontology (GO) terms. WEGO was utilised to classify GO function []. [...] To synthesis cDNA, total RNA from diluted stocks of the same RNA that was subjected to RNA-seq was used in each reverse transcription reaction using the ReverTra Ace qRT-PCR Kit (Toyobo, Japan). qRT-PCR was performed using SYBR-Green chemistry and the iCycler iQ™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) []. The primers used to amplify the targeted genes were designed using Primer Premier 5.0 (Additional file : Table S). Melting curve analysis of the PCR products was conducted at the end of each PCR cycle to verify the amplicon specificity. The mRNA expression levels of the target genes were normalized relative to the expression of the housekeeping gene actin2 to minimise sample variation. All qRT-PCR reactions were repeated with three independent biological replicates and two technical replicates. The data were analysed based on the method of Livak and Schmittgen []. […]

Pipeline specifications

Software tools Bowtie, Blast2GO, WEGO, Primer Premier
Databases SRA
Application RNA-seq analysis
Organisms Solanum lycopersicum
Diseases Infection, Mycoses
Chemicals Calcium, Salicylic Acid