RNA-Seq rhizosphere
DNA was extracted from the rhizosphere using the innuSPEED Soil DNA Kit 2.0 (LOT # 004-23) following the manufacturers instructions. DNA concentration was determined by NanoDrop. Purity and completeness were evaluated using 1% agarose gel electrophoresis. The 16S rDNA genes were amplified using the specific primers for V3 and V4 hypervariable regions; 341F (CCTAYGGGRBGCASCAG) and 806R (GGACTCANNGGGTATCTAAT). PCR was performed in a 30 L reaction with 15 L Master Mix (New England Biolabs), 0.2 M each of forward and reverse primers and 10 ng of template DNA. The thermocycling conditions were as follows: predegeneration at 98 for 1 min, 30 cycles of denaturation at 98 C for 10 s, annealing at 50 C for 30 s, and extension at 72 for 30 s, followed by a final extension at 72 for 5 min. Electrophoretic detection was performed on a 2% agarose gel. Samples with a bright main band between 400 and 450 bp were selected for subsequent experiments. PCR products were purified using Kit (Tiangen Biotech). The purified products were used to prepare the library. Sequencing libraries were generated using TIANSeq Fast DNA Library Prep Kit (Tiangen Biotech). Library quality was assessed on a Qubit 2.0 fluorometer (Thermo Scientific) and an Agilent 2100 Bioanalyzer. Finally, the libraries were sequenced on the Illumina platform using a 2250 bp paired-end protocol