title: RNA-seq of Robinia pseudoacacia tissues root, stem, leaf and nodule under control conditions and after inoculation with rhizobia strain Mesorhizobium huakuii QD9
design: RNA-seq of Robinia pseudoacacia tissues root, stem, leaf and nodule under control conditions and after inoculation with rhizobia strain Mesorhizobium huakuii QD9
library_construction_protocol: Plant tissue sampling was carried out after 5.5-months of seedlings cultivation and 3 weeks (21 days) post rhizobia-inoculation (dpi) in greenhouse. Firstly, the whole seedlings were taken out carefully from the pots to prevent any damage of the roots and washed several times with distilled water. The leaves, stem, roots and nodules were dissected, transferred separately to 10 ml centrifuge tubes, immediately frozen in liquid N2, transferred to the laboratory, ground to a fine powder under liquid N2 with a freezer mill (Spex, Metuchen, USA) and stored in a freezer at -80 °C until total RNA isolation and analysis. Robinia seedlings used for the present investigations were cultivated in a greenhouse from July to September. The average temperature in the greenhouse was 25 - 35°C and artificial illumination was applied for 12 h at 500 µE m-2 s-1 photosynthetic active radiation (PAR) during cultivation. From the 01st, July to the 01st, October, 250 ml full nutrient solution (0.05 mM K2SO4, 0.5 mM KH2PO4, 0.25 mM MgSO4·7H2O, 1 mM CaCl2, 0.01 mM Ferric citrate, 0.3 mM KNO3, 0.1 mM NH4NO3, 2 μM H3BO3, 1 μM MnSO4·H2O, 0.2 μM CuSO4·5H2O, 0.1 μM CoSO4·7H2O, 0.5 μM ZnSO4·7H2O, 0.1 μM Na2MoO4·2H2O) per pot every 14 days and 250 ml deionized H2O per pot every 3-5 days were required for normal seedlings growth and applied without seepage to avoid leaching of nutrients from the bottom of the pots. The watering scheme of 250 ml deionized H2O per pot every 3-5 days and the P concentration of 0.5 mM was required for sufficient water supply and P nutrition for plant growth. The N concentration of 0.5 mM was adapted from a previous N supply experiment of Robinia seedlings grown in a greenhouse and comparable with the soil conditions on the Loess Plateau in northwest China. Seedlings of similar size and height were selected for the subsequent inoculation experiment. The roots of half of the Robinia seedlings were inoculated with rhizobia strain Mesorhizobium huakuii QD9. For inoculation, the isolated rhizobia strain of M. huakuii QD9 was firstly cultivated on YMA solid medium (mannitol 10 g L−1, yeast extract 0.12 g L−1, NaCl 1 g L−1, MgSO4 0.2 g L−1, K2HPO4 0.5 g L−1 and agar 20 g L−1; pH=6.8) and then transferred into TY liquid medium (6 g of tryptone, 3 g of yeast extract, and 0.38 g of CaCl2 in 1 L of dH2O) with a sterilized bamboo stick, cultivated on a shaker at 28 °C for 48 hours, and centrifuged at 25 °C and 2,000 g for 10 min. From the pellet, a bacterial suspension with OD600=1.0 was produced. The roots of the Robinia seedlings were inoculated with 10 mL of this bacterial suspension using a sterile syringe. The first rhizobia-inoculation was performed at ca. 21 weeks after the cultivation of Robinia seedlings in greenhouse and subsequently three times every 7-10 days. Plant material was grounded in liquid nitrogen with a freezer mill (Spex, Metuchen, USA) and 100 mg was used for total RNA isolation via the “RNAeasy Plant Mini Kit” (Qiagen; Hilden, Germany). The RNA of three preparations was pooled and cleaned again by using a new preparation column. The quality and integrity of the resulted RNA was controlled by capillary gel electrophoresis on an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies; Waldbronn, Germany) to determine the RNA integrity number. The RNA sequencing library was generated from 1 µg total RNA using the “Ultra II Directional-RNA Seq Library-Kit” (NEB, Ipswich, USA).