title: 16s amplicon sequencing  of control rat faeces rep4
design: Majorbio (Shanghai, China) performed 16S amplicon sequencing using the Illumina MiSeq platform. DNA was extracted from rats faeces by using conventional methods. DNA concentration and quality were quantified using a Qubit fluorometer (ThermoFisher, USA). The V3-V4 regions of the 16S rRNA gene were amplified using gene-specific primers with Illumina adapter overhangs. PCR amplification was performed using a reaction mix containing DNA, KAPA HiFi HotStart Ready Mix, and primers, followed by cleanup with magnetic beads. A second PCR was performed to incorporate barcodes and sequencing adapters. The final library was normalized and pooled, then sequenced on the MiSeq (Illumina, San Diego, CA) using a 600 cycle (2 x 300 bp paired-end) run.