RNAseq of Solanum lycopersicum: leaf tissue
A total of six groups of RNA samples (drought, temperature, CPB damage, P. syringae infection, B. cinerea infection and Hexanoic acid treatment), each with three biological replicates and the corresponding control for each group were prepared. A total of 36 total RNA samples were used to construct sRNA libraries. Small RNA fraction was enriched using the Pure Link miRNA Isolation kit (Invitrogen). The amount and quality of miRNA in the samples was assessed with the Agilent 2100 Bioanalyzer using the Small RNA Chip Kit (Agilent). Small RNA libraries were constructed using the SOLID Total RNA-Seq Kit (Life Technologies). Ligation of the sequencing adapters was performed directly to the small RNA, followed by reverse transcription to cDNA. In this way, strand orientation is preserved and the strand a miRNA derives from can be exactly mapped. Bar-codes were used to prepare the libraries to sequence both strands simultaneously in a single lane. After cDNA library preparation, samples were pooled and single molecule clonal amplification of all libraries was done by emulsion PCR. For the emulsion PCR, SOLID EZ beads System was used. The amplified beads were enriched and loaded in 6 lanes on the 5500xl Genetic Analyzer system (Applied Biosystems™). Sequencing of 50 bases was performed using the ECC (Exact Call Chemistry) module (Life Technologies).