ERS6470213 SAMEA8785852 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785852 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Nodule_1d_1 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root nodule sample name E-MTAB-10497:PEG_Nodule_1d_1 ERS6470214 SAMEA8785853 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785853 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Nodule_1d_2 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root nodule sample name E-MTAB-10497:PEG_Nodule_1d_2 ERS6470215 SAMEA8785854 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785854 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Nodule_1d_3 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root nodule sample name E-MTAB-10497:PEG_Nodule_1d_3 ERS6470216 SAMEA8785855 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785855 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Nodule_3d_1 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root nodule sample name E-MTAB-10497:PEG_Nodule_3d_1 ERS6470217 SAMEA8785856 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785856 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Nodule_3d_2 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root nodule sample name E-MTAB-10497:PEG_Nodule_3d_2 ERS6470218 SAMEA8785857 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785857 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Nodule_3d_3 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root nodule sample name E-MTAB-10497:PEG_Nodule_3d_3 ERS6470219 SAMEA8785858 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785858 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Nodule_5d_1 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root nodule sample name E-MTAB-10497:PEG_Nodule_5d_1 ERS6470220 SAMEA8785859 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785859 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Nodule_5d_2 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root nodule sample name E-MTAB-10497:PEG_Nodule_5d_2 ERS6470221 SAMEA8785860 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785860 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Nodule_5d_3 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root nodule sample name E-MTAB-10497:PEG_Nodule_5d_3 ERS6470222 SAMEA8785861 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785861 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Nodule_6h_1 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root nodule sample name E-MTAB-10497:PEG_Nodule_6h_1 ERS6470223 SAMEA8785862 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785862 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Nodule_6h_2 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root nodule sample name E-MTAB-10497:PEG_Nodule_6h_2 ERS6470224 SAMEA8785863 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785863 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Nodule_6h_3 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root nodule sample name E-MTAB-10497:PEG_Nodule_6h_3 ERS6470225 SAMEA8785864 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785864 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Nodule_Control_1 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root nodule sample name E-MTAB-10497:PEG_Nodule_Control_1 ERS6470226 SAMEA8785865 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785865 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Nodule_Control_2 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root nodule sample name E-MTAB-10497:PEG_Nodule_Control_2 ERS6470227 SAMEA8785866 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785866 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Nodule_Control_3 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root nodule sample name E-MTAB-10497:PEG_Nodule_Control_3 ERS6470228 SAMEA8785867 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785867 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Root_1d_1 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root sample name E-MTAB-10497:PEG_Root_1d_1 ERS6470229 SAMEA8785868 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785868 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Root_1d_2 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root sample name E-MTAB-10497:PEG_Root_1d_2 ERS6470230 SAMEA8785869 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785869 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Root_1d_3 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root sample name E-MTAB-10497:PEG_Root_1d_3 ERS6470231 SAMEA8785870 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785870 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Root_3d_1 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root sample name E-MTAB-10497:PEG_Root_3d_1 ERS6470232 SAMEA8785871 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785871 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Root_3d_2 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root sample name E-MTAB-10497:PEG_Root_3d_2 ERS6470233 SAMEA8785872 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785872 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Root_3d_3 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root sample name E-MTAB-10497:PEG_Root_3d_3 ERS6470234 SAMEA8785873 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785873 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Root_5d_1 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root sample name E-MTAB-10497:PEG_Root_5d_1 ERS6470235 SAMEA8785874 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785874 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Root_5d_2 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root sample name E-MTAB-10497:PEG_Root_5d_2 ERS6470236 SAMEA8785875 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785875 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Root_5d_3 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root sample name E-MTAB-10497:PEG_Root_5d_3 ERS6470237 SAMEA8785876 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785876 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Root_6h_1 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root sample name E-MTAB-10497:PEG_Root_6h_1 ERS6470238 SAMEA8785877 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785877 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Root_6h_2 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root sample name E-MTAB-10497:PEG_Root_6h_2 ERS6470239 SAMEA8785878 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785878 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Root_6h_3 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root sample name E-MTAB-10497:PEG_Root_6h_3 ERS6470240 SAMEA8785879 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785879 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Root_Control_1 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root sample name E-MTAB-10497:PEG_Root_Control_1 ERS6470241 SAMEA8785880 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785880 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Root_Control_2 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root sample name E-MTAB-10497:PEG_Root_Control_2 ERS6470242 SAMEA8785881 3880 Medicago truncatula Protocols: 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). ENA first public 2021-11-30 ENA last update 2021-11-30 External Id SAMEA8785881 INSDC center alias Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC center name Institut national de recherche en agriculture, alimentation et environnement (INRAE) INSDC first public 2021-11-30T00:19:07Z INSDC last update 2021-11-30T00:19:07Z INSDC status public Submitter Id E-MTAB-10497:PEG_Root_Control_3 age 45 broker name ArrayExpress common name barrel medic cultivar Jemalong developmental stage sporophyte vegetative stage genotype A17 growth condition Sinorhizobium medicae md4 inoculation organism part root sample name E-MTAB-10497:PEG_Root_Control_3