alias: E-MTAB-8597:DN_1d_1_s
title: Illumina HiSeq 2500 sequencing; Time-course transcriptome analysis of mature nodules in Medicago truncatula grown in split-root under different nitrogen (N) supply
design: Time-course transcriptome analysis of mature nodules in Medicago truncatula grown in split-root under different nitrogen (N) supply
library_construction_protocol: We collected three replicate samples, for each time point, and each replicate was a pool of nodules coming from a single plant. For the transcriptome experiments, samples were collected at time 0 (controls) and after 1 and 3 days after the change of nutrient solution. Seeds of Medicago truncatula genotype A17 were scarified in H2SO4 97% for 5 minutes, cold-treated at 4.0°C in water for 48 hours, before germination at room temperature in the dark. After four days, the primary root tips were cut to promote branching of the root system. Individual plantlets were transferred onto hydroponic culture tanks containing a vigorously aerated basal nutrient solution renewed every week containing KH2PO4 1mM, MgSO4 1mM, 0.25mM CaCl2, 0.25mM K2SO4, 50μM KCl, 30μM H3BO3, 5μM MnSO4, 1μM ZnSO4, 1μM CuSO4, 0.7μM (NH4)6Mo7O24, 100μM Na-Fe-EDTA supplemented with 1mM KNO3. The pH was adjusted to 5.8 with KOH. Plants were grown under the following environmental parameters: 16 hours light / 8 hours dark cycles, 250 μmol s−1 m−2 photosynthetically active radiation light intensity, 22°C/20°C day/night temperature, and 70% hygrometry. The 3-week-old plants were transferred to a nutrient solution adjusted to pH7 supplemented with KNO3 0.5 mM containing the Sinorhizobium medicae md4 bacteria (107 cfu/ml). Typically, nodules appeared after 4 to 6 days and were functional after two weeks. After inoculation, nutrients solutions, renewed every week, were adjusted to pH7 but not supplemented with mineral N.   For split-root experiments, the root systems of 5-week-old plants were separated into two parts, each side being installed in a separate compartment filled with the same aerated nutrient solution. After four days, differential N treatments were initiated that modify the N provision to one side of the root system while the other side remains supplied with an aerated nutrient solution without mineral N. The N-satiety treatment (SN plants) consists in supplying the roots with a nutrient solution supplemented with 10 mM NH4NO3 . The N-limitation treatment (DN plants) was achieved by removing N2 from the treated compartment by applying a continuous flow of 80% argon / 20% O2.  Nutrient solutions were changed daily after the initiation of treatments.  At first grinding with mortar and pestle is carried out in liquid nitrogen to allow to homogenise the whole sample. We took an aliquot of 200mg for the extraction of RNAs using miRNeasy® Mini Kit (Qiagen, Venlo, Netherlands) according to the supplier's recommendations. RNAs concentration were measured using the NANODROP 1000 spectrometer (Thermo-Fisher Scientific, Waltham, Massachusetts, USA) and samples kept at -20°C. RNA integrity quality control was assessed using the BioAnalyser (BioRad Laboratories Inc., Hercules, California, USA). For each RNA extraction polyadenylated plant mRNA libraries were generated and sequenced. Library preparation and sequencing were performed at the MGX-Montpellier GenomiX Facilities (Montpellier, France). Libraries for sequencing were prepared using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, USA). Library quality was checked using the Standard Sensitivity NGS Kit on the Advanced Analytical Fragment Analyzer (Agilent Technologies, Inc., Santa Clara, California, USA), and concentration optimised performing a qPCR (Light Cycler 480, Roche, Basel, Switzerland).
identifiers.namespace: Institut National de la Recherche Agronomique (INRA)
identifiers.id: E-MTAB-8597:DN_1d_1_s
attributes.tag: Experimental Factor: growth condition
attributes.value: N-limitation
attributes.tag: Experimental Factor: time
attributes.value: 1