ITS sequencing of rhizosphere microbiome: Astragalus root soil samples
PCR amplification was carried out with an extension at 72 C for 100 seconds. After 30 cycles, a final extension step was performed at 72 C for 10 minutes, and the amplified product was stored at 4 C. Following PCR, the amplicons were subjected to 1.8% agarose gel electrophoresis. Target fragments were excised and purified using the Qiagen Gel Extraction Kit. The sequencing library was then constructed using the Illumina TruSeq Nano DNA LT Library Prep Kit, following the manufacturers protocol. Library quality was assessed with the Agilent High Sensitivity DNA Kit and the Quant-iT PicoGreen dsDNA Assay Kit. Finally, paired-end sequencing was performed on the Illumina MiSeq v3 platform (2  300 bp).