GSM9032646: 1-4; Mus musculus; OTHER
For RNA sequencing, after extracting total RNA from the samples, eukaryotic mRNA was enriched using magnetic beads coated with Oligo(dT). For 16s rRNA sequencing, Total fecal bacterial DNA was extracted using a QIAamp® Fast DNA Stool Mini Kit (QIAamp, Germany). For RNA sequencing, the mRNA was fragmented into short pieces by adding fragmentation buffer. Using the fragmented mRNA as a template, the first strand of cDNA was synthesized with random hexamer primers. Subsequently, the second cDNA strand was synthesized by adding buffer, dNTPs, RNase H, and DNA polymerase I. The double-stranded cDNA then underwent end repair, poly(A) tailing, and sequencing adapter ligation. The product was purified and size-selected using magnetic beads. Finally, PCR amplification was performed to generate the sequencing library. 16s rRNA sequencing: The 16S rRNA gene sequencing procedure was carried out by the Bioyigene Biotechnology Co. Ltd (Wuhan, China) using an Illumina HiSeq platform. We used the Thermo Scientific NanoDrop 2000 UV microspectrophotometer and 1.2 % agarose gel electrophoresis for quality control. Specific primers (341F: 5'-ACTCCTACGGGRSGCAGCAG-3', 806R: 5'-GGACTACVVGGGTATCTAATC-3') were prepared for PCR amplification using TransStart® FastPfu high fidelity DNA polymerase, and product purification was finished by Vazyme VAHTSTM DNA Clean Beads. First, the sequencing library was constructed and underwent quality inspection. Next, the libraries were sequenced using Illumina Novaseq. The library sequences were quality filtered, trimmed, denoised, and merged, and then the chimeric sequences were identified and removed to analyze the abundance, diversity and composition of the gut microbiota.