Total DNA was extracted from stool samples of 20 mice using the QIAamp®DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocols. DNA extracts were determined by agarose gel electrophoresis (1% w/v agarose) and quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).
The V3-V4 region of the 16S rRNA gene was amplified by PCR using a 30 μl mixture containing 0.5 μl of DMSO, 1.0 μl of 319F (10 mM), 1.0 μl of 806R (10 mM), 5.0 μl of the DNA sample, 7.5 μl of ddH2O, and 15.0 μl of Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB) (He et al., 2016 (link)). The reactions were hot-started at 98°C for 30 s, followed by 30 cycles of 98°C for 15 s, 58°C for 15 s, and 72°C for 15 s, with a final extension step at 72°C for 1 min. Subsequently, the amplicons were purified according to standard procedures, quantified, pooled and sequenced with the MiSeq Reagent Kits v3 (600 cycles, Illumina) according to the manufacturer’s instructions with 20% OhiX (Illumina). The sequencing reaction was conducted by Hangzhou Guhe Information and Technology Co., Ltd., Zhejiang, China.
The V3-V4 region of the 16S rRNA gene was amplified by PCR using a 30 μl mixture containing 0.5 μl of DMSO, 1.0 μl of 319F (10 mM), 1.0 μl of 806R (10 mM), 5.0 μl of the DNA sample, 7.5 μl of ddH2O, and 15.0 μl of Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB) (He et al., 2016 (link)). The reactions were hot-started at 98°C for 30 s, followed by 30 cycles of 98°C for 15 s, 58°C for 15 s, and 72°C for 15 s, with a final extension step at 72°C for 1 min. Subsequently, the amplicons were purified according to standard procedures, quantified, pooled and sequenced with the MiSeq Reagent Kits v3 (600 cycles, Illumina) according to the manufacturer’s instructions with 20% OhiX (Illumina). The sequencing reaction was conducted by Hangzhou Guhe Information and Technology Co., Ltd., Zhejiang, China.
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