ERX4820769 E-MTAB-9932:LN_0dpi_1_s ERP126011 PRJEB42176 Time-course transcriptome analysis in roots of N-limited Medicago truncatula plants grown in split-root inoculated with Sinorhizobium medicae ERS5488525 SAMEA7741171 LN_0dpi_1_s RNA-Seq TRANSCRIPTOMIC Oligo-dT 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. 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, 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 at 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 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). Illumina HiSeq 2500 Experimental Factor: infect none Experimental Factor: time 0 ERX4820770 E-MTAB-9932:LN_0dpi_2_s ERP126011 PRJEB42176 Time-course transcriptome analysis in roots of N-limited Medicago truncatula plants grown in split-root inoculated with Sinorhizobium medicae ERS5488526 SAMEA7741172 LN_0dpi_2_s RNA-Seq TRANSCRIPTOMIC Oligo-dT 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. 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, 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 at 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 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). Illumina HiSeq 2500 Experimental Factor: infect none Experimental Factor: time 0 ERX4820771 E-MTAB-9932:LN_0dpi_3_s ERP126011 PRJEB42176 Time-course transcriptome analysis in roots of N-limited Medicago truncatula plants grown in split-root inoculated with Sinorhizobium medicae ERS5488527 SAMEA7741173 LN_0dpi_3_s RNA-Seq TRANSCRIPTOMIC Oligo-dT 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. 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, 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 at 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 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). Illumina HiSeq 2500 Experimental Factor: infect none Experimental Factor: time 0 ERX4820772 E-MTAB-9932:LN_1dpi_1_s ERP126011 PRJEB42176 Time-course transcriptome analysis in roots of N-limited Medicago truncatula plants grown in split-root inoculated with Sinorhizobium medicae ERS5488528 SAMEA7741174 LN_1dpi_1_s RNA-Seq TRANSCRIPTOMIC Oligo-dT 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. 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, 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 at 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 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). Illumina HiSeq 2500 Experimental Factor: infect Sinorhizobium medicae Experimental Factor: time 1 ERX4820773 E-MTAB-9932:LN_1dpi_2_s ERP126011 PRJEB42176 Time-course transcriptome analysis in roots of N-limited Medicago truncatula plants grown in split-root inoculated with Sinorhizobium medicae ERS5488529 SAMEA7741175 LN_1dpi_2_s RNA-Seq TRANSCRIPTOMIC Oligo-dT 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. 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, 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 at 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 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). Illumina HiSeq 2500 Experimental Factor: infect Sinorhizobium medicae Experimental Factor: time 1 ERX4820774 E-MTAB-9932:LN_1dpi_3_s ERP126011 PRJEB42176 Time-course transcriptome analysis in roots of N-limited Medicago truncatula plants grown in split-root inoculated with Sinorhizobium medicae ERS5488530 SAMEA7741176 LN_1dpi_3_s RNA-Seq TRANSCRIPTOMIC Oligo-dT 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. 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, 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 at 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 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). Illumina HiSeq 2500 Experimental Factor: infect Sinorhizobium medicae Experimental Factor: time 1 ERX4820775 E-MTAB-9932:LN_2dpi_1_s ERP126011 PRJEB42176 Time-course transcriptome analysis in roots of N-limited Medicago truncatula plants grown in split-root inoculated with Sinorhizobium medicae ERS5488531 SAMEA7741177 LN_2dpi_1_s RNA-Seq TRANSCRIPTOMIC Oligo-dT 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. 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, 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 at 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 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). Illumina HiSeq 2500 Experimental Factor: infect Sinorhizobium medicae Experimental Factor: time 2 ERX4820776 E-MTAB-9932:LN_2dpi_2_s ERP126011 PRJEB42176 Time-course transcriptome analysis in roots of N-limited Medicago truncatula plants grown in split-root inoculated with Sinorhizobium medicae ERS5488532 SAMEA7741178 LN_2dpi_2_s RNA-Seq TRANSCRIPTOMIC Oligo-dT 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. 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, 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 at 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 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). Illumina HiSeq 2500 Experimental Factor: infect Sinorhizobium medicae Experimental Factor: time 2 ERX4820777 E-MTAB-9932:LN_2dpi_3_s ERP126011 PRJEB42176 Time-course transcriptome analysis in roots of N-limited Medicago truncatula plants grown in split-root inoculated with Sinorhizobium medicae ERS5488533 SAMEA7741179 LN_2dpi_3_s RNA-Seq TRANSCRIPTOMIC Oligo-dT 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. 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, 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 at 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 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). Illumina HiSeq 2500 Experimental Factor: infect Sinorhizobium medicae Experimental Factor: time 2 ERX4820778 E-MTAB-9932:LN_7dpi_1_s ERP126011 PRJEB42176 Time-course transcriptome analysis in roots of N-limited Medicago truncatula plants grown in split-root inoculated with Sinorhizobium medicae ERS5488534 SAMEA7741180 LN_7dpi_1_s RNA-Seq TRANSCRIPTOMIC Oligo-dT 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. 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, 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 at 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 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). Illumina HiSeq 2500 Experimental Factor: infect Sinorhizobium medicae Experimental Factor: time 7 ERX4820779 E-MTAB-9932:LN_7dpi_2_s ERP126011 PRJEB42176 Time-course transcriptome analysis in roots of N-limited Medicago truncatula plants grown in split-root inoculated with Sinorhizobium medicae ERS5488535 SAMEA7741181 LN_7dpi_2_s RNA-Seq TRANSCRIPTOMIC Oligo-dT 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. 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, 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 at 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 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). Illumina HiSeq 2500 Experimental Factor: infect Sinorhizobium medicae Experimental Factor: time 7 ERX4820780 E-MTAB-9932:LN_7dpi_3_s ERP126011 PRJEB42176 Time-course transcriptome analysis in roots of N-limited Medicago truncatula plants grown in split-root inoculated with Sinorhizobium medicae ERS5488536 SAMEA7741182 LN_7dpi_3_s RNA-Seq TRANSCRIPTOMIC Oligo-dT 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. 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, 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 at 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 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). Illumina HiSeq 2500 Experimental Factor: infect Sinorhizobium medicae Experimental Factor: time 7