title: RNA-Seq of mouse D2
design: The faeces were collected and the DNA was extracted using a DNeasy Power Soil kit in accordance with the manufacturer's instructions.Using the CTAB/SDS technique, total genomic DNA was extracted from the chosen samples. By loading on 1% agarose gel electrophoresis, DNA concentration and purity were verified. DNA was diluted in sterile water to 1 ng/ l. Then, specific primer with a barcode was used to amplify 16S rRNA. The Phusion High-Fidelity PCR Master Mix (New England Bio-Labs), 0.2 M forward and reverse primers, and roughly 10 ng of template DNA were added to a 30 l reaction system to run the PCR. Thermal cycling was designed to run for 30 cycles of initial denaturation at 98C for 10 s, annealing at 50C for 30 s, elongation at 72C for 60 s, and 72C for 5 min. PCR products were combined with 1  volume of loading buffer (which contained SYB green) and electrophoresed on a 2% agarose gel for detection. For further experiments, the samples with a brighter strip between 400 and 450 bp were chosen. Equidensity ratios were used to mix the PCR products. With the help of a Gene JET gel extraction kit from Thermo Scientific, the PCR product mixture was further purified. According to Li et al.'s instructions, library construction, sequencing, and bioinformatics analysis were completed