title: RNA-Seq of mouse: stool MP1000-1
design: Microbial DNA was isolated from frozen appendicular fecal samples using the QIAamp DNA Feces Mini Kit (Qiagen, Venlo, Netherlands), in accordance with the manufacturer's protocols. Subsequently, the V3-V4 region of the 16S rRNA gene was amplified from the DNA using a bacterial universal primer set (341F and 806R). PCR was conducted using EF-Taq (Korea, Solgent), with 20 ng of genomic DNA serving as the template in a 30 L reaction mixture. The thermocycling parameters were as follows: initial activation of Taq polymerase at 95 C for 2 minutes, followed by 35 cycles of denaturation at 95 C, annealing at 55 C, and elongation at 72 C for 1 minute each, and a final extension step at 72 C for 10 minutes. The PCR products were purified on a multiscreen filter plate (Millipore Corp., Billerica, MA, USA).