mouse gut metagenome Metagenome

Eight-week-old male C57BL/6J mice were randomly assigned to five groups: (1) control diet (Research Diets, Inc., NJ, USA; D12450K); (2) P-HFD (Gubra-Amylin NASH (GAN) diet; Research Diets, Inc., NJ, USA; D09100310) + intraperitoneal injections of LPS (500 ug/kg bw/week; LPS from Escherichia coli O55:B5; Sigma-Aldrich; L2880) (PL); (3) PL + a low dose of GEO (12.5 mg/kg bw/day) (PL+GL); (4) GAN/LPS + a medium dose of GEO (62.5 mg/kg bw/day) (PL+GM); and (5) GAN/LPS + a high dose of GEO (125 mg/kg bw/day) (PL+GH). The mice were sacrificed by carbon dioxide asphyxiation after 12 weeks. Mouse fecal samples were removed from the colons during the sacrifices, snap-frozen using liquid N2, and stored at -80 C before use. Genomic DNA from the feces was extracted using the QIAmp Power Fecal Pro DNA Kit (QIAGEN, Netherlands) and quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). The V3-V4 hypervariable region of the 16S rRNA gene was amplified using the primer pair ((Forward=5-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3) and Reverse=5-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3)). Polymerase chain reaction (PCR) amplification was conducted in a 25 uL reaction mixture containing 5 ng of DNA template, 0.2 uM forward and reverse primers, and 12.5 uL of 2xTaq Master Mix (KAPA HiFi HotStart ReadyMix, Roche, Switzerland). The PCR conditions included an initial step at 95 C for 3 min, followed by 25 cycles of 95 C, 55 C, and 72 C for 30 s each, and a final extension at 72 C for 5 min. Amplified products were subsequently visualized by using 2% agarose gel electrophoresis. Dual index and Illumina sequencing adapters were joined using a Nextera XT Index Kit via PCR. PCR product cleanup was conducted using AMPure XP beads to purify amplicon. The sizes of PCR products were validated using the Bioanalyzer DNA 1000 chip. Library quantification was carried out for quality control before sequencing using the Agilent Technologies 2100 Bioanalyzer. The pooled libraries were subjected to paired-end sequencing (2x300 bps) using the Illumina MiSeq platform.