NextSeq 500 sequencing; Dynamic transcriptome response of rice roots and shoots after treatment with different forms of nitrogen
Rice accessions (Oryza sativa Japonica cv. Nipponbare) were obtained from the USDA-ARS, Dale Bumpers National Rice Research Center, Stuttgart, Arkansas, Genetic Stocks Oryza Collection (www.ars.usda.gov/GSOR). The full roots and shoot tissues were harvested separately. The seed was discarded at the moment of harvest. Rice seedlings (Oryza sativa Nipponbare cultivar) were dehulled and sterilized with ethanol 70% for 5 minutes, followed by immersion in bleach 6% with Tween-20 for 30 minutes. Seedlings were imbibed by immersion in sterile water for 12h to synchronize germination. Germinating seeds were transferred on a hydroponic system, and roots were immersed in a nitrogen-free basal salt medium composed of K2SO4 0.7mM, KH2PO4 0.3mM, CaCl2.2H2O 1mM, MgSO4.7H2O 1mM, Na2Sio03.9H2O, Na2-Fe-EDTA (20µM) for macronutrients, and MnCl2.4H2O 9µM, Na2Mo04.2H2O 0.39µM, H3BO3 20µM, ZnSO4.7H2O 0.77µM, CuSO4.5H2O 0.32µM for micronutrients (pH 5.8). Seedlings were then transferred to a growth cabinet in the dark at 30 degrees for 3 days in a randomized block design. The light was then turned on after 72h. Nitrogen treatments consisted of injection with KNO3 (5mM NO3- treatment), 2.5mM (NH4)2SO4 + 2.5mM K2SO4 (5mM NH4+ treatment), 2.5mM KNO3 + 1.25mM (NH4)2SO4 (NH4NO3 treatment) or 2.5mM K2SO4 (negative control) in the basal medium. Rice seedlings were extracted 15min, 1h, 2h, 4h, 12h, 24h and 48h after nitrogen treatments. At the extraction time-point, shoots and roots were cut with a razor blade and frozen in liquid nitrogen. The remaining seeds were discarded. A 0h time-point was also harvested to control sampling effects and used in comparison to the Mock 0h time point in our analyses. Three different boxes were used for each treatment and for each time-point, for a total of 87 boxes and at least 10 germinated seedlings were sampled per box. Frozen roots and shoot samples were grinded with one 3mm metal bead into Eppendorf tubes. RNA was extracted with Trizol (Life Technologies) and the RNeasy Mini Kit (Qiagen) following the manufacturer instructions. An extra DNase step was performed with RNase-Free DNase Set (Qiagen). RNA samples were resuspended in RNAse free water. RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). The sequencing and library preparation was performed by the VIB Nucleomics Core Facility (Leuven, Belgium / www.nucleomics.be). Per sample, 500ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version: Part # 15031047 Rev. E - October 2013), 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 PolymeraseI 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 conducted to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library.