Genomic DNA of each sample was extracted with the Plant DNA Extraction Kit (Tiangen, Beijing, China) according to the manufacturer’s protocols. Total DNA was eluted with 50 μL TE buffer (Tris-hydrochloride buffer, pH 8.0, containing 1.0 mM EDTA). The DNA concentration and purity were determined spectroscopically using NanoDropTM 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), then stored at −80 °C until PCR amplification.
The V3-V4 region of bacterial 16S ribosomal RNA gene was PCR-amplified (98 °C for 30 s; followed by 35 cycles at 98 °C for 10 s, 54 °C for 30 s, and 72 °C for 45 s; and a final extension at 72 °C for 10 min) using a primer set of 341F (5’-CCTACGGGNGGCWGCAG-3’) and 805R (5’-GACTACHVGGGTATCTAATCC-3’) [27 (link)]. The 5′ ends of the primers were tagged with specific barcodes per sample and sequenced using universal primers. All PCR reactions were conducted in a 25 μL mixture containing 12.5 μL of 2× Phusion® Hot Start Flex Master Mix, 2.5 μL of each primer (1 μM), and 50 ng of template DNA. Nuclease-free water served as blank.
The V3-V4 region of bacterial 16S ribosomal RNA gene was PCR-amplified (98 °C for 30 s; followed by 35 cycles at 98 °C for 10 s, 54 °C for 30 s, and 72 °C for 45 s; and a final extension at 72 °C for 10 min) using a primer set of 341F (5’-CCTACGGGNGGCWGCAG-3’) and 805R (5’-GACTACHVGGGTATCTAATCC-3’) [27 (link)]. The 5′ ends of the primers were tagged with specific barcodes per sample and sequenced using universal primers. All PCR reactions were conducted in a 25 μL mixture containing 12.5 μL of 2× Phusion® Hot Start Flex Master Mix, 2.5 μL of each primer (1 μM), and 50 ng of template DNA. Nuclease-free water served as blank.
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