title: RNA-Seq Brassica napus cotyledon 50 M sirodesmin PL 16 h, replicate 3
design: Plant treatments were carried out on cotyledons of Brassica napus, using one cotyledon per plant that was detached using a razor blade. Each cotyledon was immediately transferred to a Petri dish containing 0.8% w/v agarwater. To avoid dehydration, the petioles of the cotyledons were carefully immersed in the agar. In all cases, a 10 l drop of 50 M sirodesmin PL solution (dissolved in 1% v/v ethanol) or 1% v/v ethanol (control) was applied to a lobe of each cotyledon. Then, a small puncture was made with a hypodermic needle where the drop was placed to facilitate the entry of the toxin. Finally, dishes were sealed and returned to the original growing conditions. For total RNA isolation, each biological replicate consisted of 10 cotyledon discs from different plants. Each cotyledon disc (10 mm in diameter) consisted of healthy and damaged tissue. Total RNA from control and treated samples was extracted after treatment using a Plant Spectrum Total RNA Kit (Sigma), according to the manufacturers instructions. Samples were treated with the Ambion Turbo RNA-free DNase kit according to the manufacturers protocol. Quantity and purity of total RNA were assessed spectrophotometrically in a multiplate reader Synergy H1 (BioTek). Libraries and RNA sequencing were performed by Macrogen Company (Seoul, Korea) using Illumina technology. Total RNA integrity was checked using an Agilent Technologies 2100 Bioanalyzer with an RNA integrity number (RIN) value 7. Paired-end sequencing was performed with 101 bp read length.