BKSBY1.5 (CRX1877781)
Soil samples DNA were extracted using a soil DNA extraction kit. DNA purity and concentration were monitored on 1% agarose gel electrophoresis. The V5-V7 hypervariable regions of the bacterial 16S rRNA gene were amplified with primers 799F and 1193R. Fungal ITS sequences were amplified using primers ITS5-1737F and ITS2-2043R. PCR reactions contained 15 uL of Phusion High-Fidelity PCR Master Mix (New England Biolabs), 0.2 uM of forward and reverse primers, and about 10 ng of template DNA. An equal volume of 1X loading buffer (containing SYBR green) was mixed with PCR products and run on 2% agarose gel for detection. PCR products were mixed in equimolar ratios, then purified. Sequencing libraries were generated using the TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturer's recommendations, and indexes were added. The library was quantified with Qubit and real-time PCR, and sequenced on the Illumina NovaSeq platform.