16S rRNA gene sequencing of soil microbiome: response to Electric Potential Treatment + Urea
Soil total DNA was extracted using the DP336-02 TIANamp Soil DNA Kit according to the manufacturers instructions. The V4-V5 region of the bacterial 16S rRNA gene was amplified using primers 515F (5-GTGYCAGCMGCCGCGGTAA-3) and 1064R (5-AYCTCACGRCACGAGCTGAC-3). PCR reactions were performed in 2.5L volumes using 10x Phusion High-Fidelity PCR Master Mix under the following conditions: initial denaturation at 98C for 2 min; followed by 35 cycles of 98C for 10 s, 55C for 30 s, and 72C for 30 s; with a final extension at 72C for 5 min. Successful amplification was confirmed by 1.5% agarose gel electrophoresis. PCR amplicons were purified using AMPure XP beads. The purified amplicons were then submitted to Biomarker Technologies Corporation (Beijing, China) for subsequent library construction using their standard Illumina dual-index paired-end protocol (e.g., based on the Illumina TruSeq DNA PCR-Free or Nano Kit) and sequenced on an Illumina NovaSeq 6000 platform.