peanut rhizosphere Treatment3 rhizobium
The samples were thawed, homogenized, and centrifuged at 10,000 rpm for 5 minutes, causing microbial cells and soil particles to settle. The supernatant was discarded, and the soil was mixed well before proceeding with DNA extraction. DNA was extracted from 1 gram of soil using the Qiagen Power Soil DNA extraction kit (Qiagen, Germany) following the manufacturer's recommendations. The extracted DNA samples were subjected to NanoDrop ND1000 spectrophotometer (Thermo Scientific, MA, USA). A NanoDrop reading of 260/280 with a value of approximately 1.8 to 2 is used to determine DNA quality. The Quick-16S Plus NGS Library Prep Kit (V3-V4), targeting the V3-V4 region of the 16S rRNA gene was used for 16S rRNA identification and amplification using PCR. Primers V13F: 5'AGAGTTTGATGMTGGCTCAG3' and V13R: 5'TTACCGCGGCMGCSGGCAC3' were used for amplification. The amplicons from each sample were purified using Ampure beads to remove unused primers, followed by an additional 8 cycles of PCR with Illumina barcoded adapters for sequencing library preparation. Libraries were purified with Ampure beads and quantitated using the Qubit dsDNA High Sensitivity assay kit. Sequencing was conducted using the Illumina MiSeq system with a 2x300PE v3 sequencing kit.