Toptaq dna polymerase kit

PCR was conducted using a TopTaq DNA polymerase kit (Transgen, China) to amplify the V3-V4 region from the bacterial 16S rDNA gene with the primer sets Primer F (Illumina adapter sequence 1 + CCTACGGGNGGCWGCAG) and Primer R (Illumina adapter sequence 2 + GACTACHVGGGTATCTAATCC). Three 1-μL aliquots from each DNA sample were used as a template for amplification according to the manufacturer’s protocols. Illumina index sequences were specifically designed to identify each DNA sample using the following barcoded primers: Fwd 5′-AATGATACGGCGACCACCGAGATCTACACXXXXXXXXACACTCTTTCCCTACACGACGCTCTTCCGATCTCTG-3′ and Rev 5′-CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC-3′, in which X indicates a unique barcode for each sample. The amplified products were checked by agarose gel electrophoresis and purified with magnetic beads. All PCR products were sequenced by MiSeq reagent kit version 3 (Illumina, San Diego, CA, USA). Similarly, detailed sequencing methods can be found in the following literature [25 (link),38 (link),39 (link),40 (link),41 (link),42 (link)].

During the polymerase chain reaction, which was conducted with a TopTaq DNA polymerase kit (Transgen, Beijing, China), the V3–V4 regions from the bacterial 16S rDNA gene were amplified with universal primer set 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) [41 (link)]. According to the manufacturer’s protocol, final PCR products were purified and quantified by Agencourt AMPureXPPCR Purification Beads (Beckman Coulter, Grants Pass, OR, USA) and Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The sequencing was performed on Illumina NovaSeq 6000 platform (2 × 250 bp) at Genesky Biotechnologies Inc. (Shanghai, China). Amplicon sequencing is the high-throughput sequencing of PCR products of specific gene segments such as 16S rDNA, 18S rDNA, and ITS [42 (link)]. Among them, the 16S rDNA sequencing used in this study is an important and widely used tool for obtaining information about microbial communities’ structures, microbial taxonomy, and community diversity [43 (link)].

Stool samples were collected from colon of rats from the second experiment and stored at −80°C before DNA isolation using the TopTaq DNA Polymerase Kit (Transgen, China). The targeted V3-V4 hypervariable region was amplified with the primer pair F: Illumina adapter sequence 1 + CCTACGGGNGGCWGCAG and primer pair R: Illumina adapter sequence 2 + GACTACHVGGGTATCTAATCC. Amplicons from all samples were multiplexed and paired-end sequenced on an Illumina Miseq at Genesky Biotechnologies Inc., Shanghai, China, according to a published dual-indexing protocol (Xie et al., 2017 (link)).