Tguide s96 magnetic stool dna kit

Fecal genomic DNA was extracted using the TGuide S96 Magnetic Stool DNA Kit (Tiangen Biotech Co., Ltd. Beijing, China), according to manufacturer’s instructions. The DNA quality was assessed using the Qubit dsDNA HS Assay Kit and Qubit 4.0 Fluorometer (Invitrogen, Oregon, United States). The special region (V3-V4) of the 16S rRNA gene in DNA samples was amplified using the general primers 338FP 5′-ACT​CCT​ACG​GGA​GGC​AGC​A-3′ and 806RP 5′-GGACTACHVGGGTWTCTAAT-3'. The reaction volume for the polymerase chain reaction (PCR) was 10 μl, including 338FP (10 μM) 0.3 μl, 806RP (10 μM) 0.3 μl, KOD FX Neo 0.2 μl, KOD FX Neo Buffer 5 μl, dNTP (2 μM) 2 μl, DNA template 25 ng, and distilled water up to 10 μl. The PCR products were further purified using Agencourt AMPure XP Beads (Beckman Coulter, Indianapolis, IN, United States) and quantified using the Qubit 4.0 Fluorometer (Invitrogen, Thermo Fisher Scientific, Oregon, United States). Then, the purified PCR products were mixed in equal amounts to construct a library. The library was sequenced on Illumina Novaseq 6000, and further bioinformatic analysis was carried out using BMKCloud (Biomarker Technologies Co., Ltd. Beijing, China).

The bacterial DNA was extracted from 33 rumen samples using the TGuide S96 Magnetic Stool DNA Kit (Tiangen Biotech (Beijing, China) Co., Ltd.). The hypervariable region V3–V4 of the bacterial 16S rRNA gene was amplified with primer pairs 338F: 5′–ACTCCTACGGGAGGCAGCA–3′ and 806R: 5′–GGACTACHVGGGTWTCTAAT–3′. The thermocycling profile used here was as follows: 95 °C for 5 min followed by 20 subsequent cycles of denaturation at 95 °C × 30 s, 50 °C × 30 s, then 72 °C × 40 s, and a final extension at 72 °C for 7 min. PCR products were checked on agarose gel and purified through the Omega DNA purification kit (Omega Inc., Norcross, GA, USA). The purified PCR products were collected, and the paired ends (2 × 250 bp) was performed on the Illumina Novaseq 6000 platform (Beijing Biomarker Technologies Co., Ltd., Beijing, China).