title: Illumina HiSeq 2500 sequencing; Transcriptional response of Arabidopsis thaliana to exogenous application of pipecolic acid
design: Transcriptional response of Arabidopsis thaliana to exogenous application of pipecolic acid
library_construction_protocol: Three biologically independent, replicate experiments were performed. In each experiment, at least 4 full-grown leaves from 6 different plants were pooled for one biological replicate per treatment. In this way, 3 biologically independent, replicate samples per treatment and plant genotype were obtained.  Arabidopsis thaliana plants were grown individually in pots containing a mixture of soil (Substrat BP3; Klasmann-Deilmann), vermiculite, and sand (8:1:1) in an enviromentally controlled cultivation chamber with a 10-h-day (9 AM to 7 PM)/14-h-night cycle. Relative humidity in the growth chambers was adjusted to 60% and day and night temperatures were set to 21°C and 18°C, respectively Experiments were performed with 5- to 6-week-old, naive plants exhibiting a uniform appearance.Treatments with pipecolic acid (Pip) were essentially performed as detailed in Návarová et al. (2012).10 ml of a 1 mM aqueous solution of Pip (equates to 10 µmol) were pipetted onto the soil of individually cultivated 5-week-old plants. 10 ml of water were applied in the control treatments. Three biologically independent, replicate experiments were performed. In each experiment, 24 hours after the watering with Pip or water, at least 4 full-grown leaves from 6 different plants were pooled for one biological replicate. RNA was extracted from 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). RNA integrity, sequencing library quality and fragment size were checked on a 2100 Bioanalyzer (Agilent, CA, USA).  Libraries were prepared using the TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA) and library quantification was performed with a Qubit 2.0 (Invitrogen, Germany). Leaf samples were multiplexed 12 libraries per lane yielding approximately 150 million reads per lane.