Illumina NovaSeq 6000 sequencing; Effect of the sunn mutation on systemic signalling of the plant N demand in Medicago truncatula plants inoculated for 2 or 7 days with Sinorhizobium medicae
Effect of the sunn mutation on systemic signalling of the plant N demand in Medicago truncatula plants inoculated for 2 or 7 days with Sinorhizobium medicae
We initiated N-treatments and inoculated plants at different time before harvest. We collected all roots samples simultaneously in SN or LN compartments of the split-root systems. Each biological replicate is a pool of the half root systems of two plants. RNAseq analysis included 3 biological replicates per condition. We collected three replicate samples, for each time point, and each replicate was a pool of roots of two plants.  The Medicago truncatula genotypes used were the wild type Jemalong A17 and the TR122 sunn mutant. 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, a 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 in two parts. Split-root compartments contained HY nutrient solution supplemented with KNO3 0.5mM as sole mineral nitrogen source and the pH was adjusted to 7. For the nitrogen treatments, one split root side was supplied with nutrient solution with mineral N, 10 mM NH4NO3 for N-satisfied (SN) plants and 0.5 mM for N-limited (LN) plants, whereas the non-N treated part was kept in nutrient solution without mineral N. Roots of all compartments were inoculated 48h after the N treatment initiation with Sinorhizobium medicae md4 (107 exponentially growing bacteria per ml). We renewed the nutrient solutions were every 4 days. We initiated N treatments at different times before harvest in order to compare plants of the same age (6 weeks old plants) differing by the duration post-inoculation. At first grinding with mortar and pestle is carried out in liquid nitrogen to allow to homogenise the 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 Rapid SBS Kit (Illumina, San Diego, California, 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).