title: Illumina HiSeq 4000 sequencing; Transcriptional response of Arabidopsis thaliana to exogenous application of N-hydroxypipecolic acid (NHP).
design: Transcriptional response of Arabidopsis thaliana to exogenous application of N-hydroxypipecolic acid (NHP).
library_construction_protocol: To generate one biological replicate sample, 18 full-grown leaves from 6 different plants (3 leaves per plant) were harvested 24 h after the respective treatment and pooled. In total, three biologically independent, replicate experiments were performed. As a result, three biologically independent samples per treatment and plant genotype were obtained. RNA-seq analysis of the resulting 21 samples was performed as described in the accompanying protocols. Individual Arabidopsis thaliana plants were cultivated in pots filled with a mixture of soil (Substrat BP3; Klasmann-Deilmann), vermiculite, and sand (8:1:1) in a controlled plant growth room. A 10-h-day (9 AM to 7 PM)/14-h-night cycle with a photon flux density of approximately 100 μmol m-2 s-1 during the day and a relative humidity of 60% was applied. Day and night temperatures were set to 21 and 18 °C, respectively. Experiments were performed with five-week old plants. To investigate the transcriptional response of Arabidopsis wild-type plants (Col-0) and selected mutants (npr1-3 and sid2-1) to elevated NHP (or Pip), individual, 5-week-old soil-grown plants, exhibiting a uniform appearance, were supplied via the root with 10 μmol NHP, 10 μmol Pip (only in the case of Col-0 plants), or treated with an equivalent volume of water, which served as solvent and thus control treatment. Total RNA was extracted from the leaf sample replicates with the Plant RNeasy extraction kit (Qiagen, Germany), and treated on-column (Qiagen, Germany) and in solution with RNA-free DNAse (New England Biolabs, MA, USA). Total RNA used for transcriptome analyses was quantified (Qubit RNA HS Assay, Thermo Fisher Scientific) and the quality measured by capillary electrophoresis using the Fragment Analyzer and the 'Total RNA Standard Sensitivity Assay' (Agilent Technologies, Inc. Santa Clara, USA). All samples in this study showed good RNA Quality Numbers (RQN; mean = 7.8).  The library preparation was performed according to the manufacturer's protocol using the 'VAHTS™ Stranded mRNA-Seq Library Prep Kit' for Illumina®. Briefly, 300 ng total RNA were used for mRNA capturing, fragmentation, the synthesis of cDNA, adapter ligation and library amplification.