RNA-seq of Saponaria officinalis tissues (leaves, stems and roots) in response to MOCK or 50 uM Methyl Jasmonate treatment at 6 and 24 hours
RNA-seq of Saponaria officinalis tissues (leaves, stems and roots) in response to MOCK or 50 uM Methyl Jasmonate treatment at 6 and 24 hours
Briefly. 3 months old Saponaria plants were harvested, using a surgical blade roots were separated from shoots and leaves, each tissue was collected separately and snap-frozen in liquid nitrogen prior processing for RNA extraction.  Seeds of Saponaria officinalis used for transcriptome sequencing were sourced from the seed company Jelitto (https://www.jelitto.com/, Schwarmstedt, Germany). Seeds were surface sterilized and placed in Magenta boxes with germination medium containing: 2.2 gr L-1 MS with vitamins (Duchefa), 1.5% sucrose, 0.1% MES, 0.4% Phytagel, pH was adjusted with 1 M KOH to 5.8. Sealed boxes were kept in the dark for 1 week at 4 degrees prior transferring to long day for germination. Seeds were germinated in long day (18 hours of light) under cold fluorescent lamps at 24 degrees. Two weeks after germination plantlets were transferred to hydroponic boxes containing nutrient solution (1/4 MS with vitamins) and grown in long day (18 hours of light) at 24 degrees for three months prior elicitation and sampling.  Elicitation was perfomed by adding Methyl Jasmonate to the nutrient solution to reach a final concentration of 50 uM, mock treated plants were instead administered with the same amount of ethanol added to the hydroponic solution. Each samples was ground in liquid nitrogen using mortar and pestle. RNA was extracted from leaves, stems and roots sampled in triplicate (each biological replicate being a pool of three individual plants) at 6 and 24 hours after treatment. RNA was extracted from the sampled S. officinalis tissues using the ReliaPrep RNA miniprep Systems (Promega™), following manufacturer's instructions for fibrous tissues. RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-8000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 500 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version: # 1000000040498 v00 October 2017) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally, enrichment PCR was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library.