Transcriptome analysis of  Medicago lupulina symbiotic roots under copper  stress
Seeds of Medicago lupulina were rubbed with sandpaper for scari?cation, sterilized with 95% ethanol for 10 min, and rinsed several times with deionized water. The seeds were subsequently germinated on 1% agar autoclaved in distilled water in the dark for 48 hours.     The germinated seeds were transferred to sterilized mix of vermiculite/perlite (volume ratio 2:1) pots without Cu or supplemented with copper sulfate (CuSO4) at the indicated concentrations, fertilized with Fåhraeus nitrogen-free nutrient solution, and cultivated in a growth chamber with a 16 h light/8 h dark cycle at 20–26 ?.   For the Cu treatment, copper sulfate solution or deionized water was added into the mix of vermiculite/perlite and equilibrated for 7 d to abtain the concentration of 0 and 200 mg/kg, respectively. Seven-d-old seedlings were inoculated with Sinorhizobium meliloti CCNWSX0020 (OD600 = 0.2).For RNA-Seq, M. lupulina roots grown under 0 (control) or 200 mg/kg Cu were collected, and two pools of total RNA were isolated from the the infected roots at 1, 2, 3, 4, 5, 6, 7, 10, 15 days after inoculation (dpi) using TRIzol Reagent (Takara, Dalian, China) according to the manufacturer’s protocol, respectively. Three biological replicate samples underwent RNA extracting and were mixed into one for each treatment.