16s-Seq of Rattus norvegicus
After DNA extraction of the samples between the different groups, primers were designed based on conserved regions of the 16S rDNA/ITS2 sequences the extracted total microbial DNA was used as a template. Phusion enzyme was employed for one-step PCR amplification using the extracted DNA as a template, with 35 cycles of amplification. Sequencing universal adaptors and sample-specific barcode sequences were added to the amplified products through PCR targeting the desired regions. The PCR products were checked by 1.5% agarose gel electrophoresis, and the target fragments were recovered using the AxyPrep PCR Cleanup Kit. Purified PCR products were quantified and pooled using the Quant-iT PicoGreen dsDNA Assay Kit on the Promega QuantiFluor fluorescence quantification system. Finally, double-end sequencing was conducted on the Illumina sequencing platform using the standard 250PE protocol.