Illumina HiSeq 2500 sequencing; Response of the mature nodules of the Medicago truncatula/sinorhizobium medicae md4 symbiotic association to systemic signaling associated to plant water deficit (PEG treatment)
We initiated PEG-treatments at different times before harvest. All samples were collected from 45 days-old plants. We collected three replicate samples for each time point, and each replicate was a pool of the nodules of 3 plants. For the transcriptome experiments, samples were collected at time 0 (controls) and after 6 hours, 1-, 3- and 5-days PEG treatment. Medicago truncatula Jemalong A17 seeds were scarified, germinated as described in Lambert et al. (Lambert, I. et al. J Exp Bot 2020 - http://dx.doi.org/10.1093/jxb/eraa221). Individual plantlets were transferred into hydroponic culture tanks containing a vigorously aerated HY basal nutrient solution adjusted to 5.8 with KOH and supplemented with 1 mM KNO3. We cut the primary root tips of plantlets to promote branching of the root system. The culture chambers conditions were a light intensity of 250 μmol s−1 m−2 photosynthetically active radiation, relative humidity of 70%, a light/dark cycle of 16h/8h and an ambient temperature of 22°C/20°C. We separated the root systems of 4-week-old plants into two parts. 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 without mineral N supplement were renewed every week and adjusted to pH7.  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. We initiated the PEG treatment (PEG 8000, 150g/l) at various times before harvest to compare plants of the same age (45 days old plants).  At first, grinding with mortar and pestle is carried out in liquid nitrogen to homogenise the sample. We took an aliquot of 200mg to extract 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).