title: Control_Root_6d_3
description: Protocols: Control and treated samples were collected at different time points (6 hours, 1-, 3- and 6-days) following the Aphanomyces euteiches. We collected three replicate samples for each time point, and each replicate was a pool of the roots of 3 plants. 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. A 50 mL suspension containing 30.000 A. euteiches zoospores /mL, produced in Volvic water as previously described (Djébali et al. MPMI, 2009 - http://dx.doi.org/10.1094/MPMI-22-9-1043), was applied only onto the local compartment on each inoculated plants that were 44 days old. At the same time, we added 50 mL of Volvic water to mock-inoculated samples (controls). 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).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-03-31T00:17:20Z
attributes.tag: individual
attributes.value: pool 21 of 3 plants
attributes.tag: organism
attributes.value: Medicago truncatula
attributes.tag: organism
attributes.value: Medicago truncatula
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-07-18T14:57:32Z
attributes.tag: scientific_name
attributes.value: Medicago truncatula
attributes.tag: common name
attributes.value: barrel medic
attributes.tag: organism part
attributes.value: root
attributes.tag: ecotype
attributes.value: Jemalong A17
attributes.tag: age
attributes.value: 50
attributes.tag: developmental stage
attributes.value: sporophyte vegetative stage
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-03-31
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-07-18