Fecal dna isolation kit

Microbial DNA was extracted from the fecal samples using the fecal DNA isolation kit (Qiagen). The abundance of the mucolytic bacteria Akkermansia, Bacteroides, and species of Bacteroides in feces was further determined by qPCR by the 2^−ΔΔCT method (53 (link), 54 (link)). The relative abundance of Akkermansia and Bacteroides was normalized to total bacteria and presented as the ratio between groups. Primers used for bacterial 16S rRNA gene detection were evaluated as previously described (6 (link), 55 (link), 56 (link)) and are listed in Table S3.

Stool samples were collected from each mouse in the 8^th week and immediately snap frozen in liquid nitrogen before storage at −80°C. The feces were collected after administration and extracted using a fecal DNA isolation kit (Qiagen, Germantown, USA). The extracted DNA from each sample was used as the template to amplify the V4 region of the 16S rRNA genes for subsequent pyrosequencing, which was performed by Novogene (Beijing, China) as described previously.^{47 (link)}

Total metagenomic DNA was extracted from fecal pellets collected from 24-week-old mice just before being sacrificed using the fecal DNA isolation kit (Qiagen, United States). The concentration and purity of DNA were estimated spectrophotometrically, and the quality was assessed by agarose gel electrophoresis. The 16S rRNA V3–V4 hyper-variable region was amplified by PCR and sequenced using Illumina Miseq PE250. After quality control of the original reads, Usearch was used for data statistics and clustering analysis: amplicon sequence variants (ASVs) were obtained by 97% similarity clustering. Each ASV was considered to represent a species. Then, according to the minimum number of sequences matched to ASV, random flattening was performed and α-diversity was analyzed. A sequence of representative read was selected from each ASV and compared with the RDP database to classify each ASV and get the species abundance table for subsequent analysis. The 16S rRNA sequence was also analyzed on the free online platform of Majorbio I-Sanger Cloud Platform (see text footnote 1).

Genomic DNA was extracted from cecal samples using the fecal DNA Isolation Kit and DNA Purification Kit according to the manufacturer's instructions (QIAGEN, Hilden, Germany). The V3–V4 region of the 16S rRNA gene was sequenced using primers 338F (5′-ACT CCT ACG GGA GGC AGC AG-3′) and 806R (5′-GGA CTA CHV GGG TWT CTA AT-3′). Sequencing of the PCR amplification products was performed at the Shanghai Major Bio-Pharm Technology (Shanghai, China) for high throughput sequencing. The raw data from high throughput sequencing that support the findings of this study are openly available at https://www.ncbi.nlm.nih.gov/genbank (Reference number: PRJNA606689).
The relative abundance of gut microbiota at operational taxonomic units (OTUs) level between the NFD and HFD groups or HFD group and SIM group were shown by an extended error bar plot using STAMP (Ver. 2.1.3). The heatmap of correlation between the intestinal microbial phylotypes of significant differences and the biochemical indexes or liver metabolites was implemented by package “pheatmap” through R software (Ver. 3.3.3). The correlation network between lipid metabolism related indexes and the key intestinal microbial phylotypes was created by Cytoscape (Ver. 3.6.0).

The total genome DNA of bacteria was extracted with a fecal DNA isolation kit (MoBio Laboratories, USA) from frozen feces according to the manufacturer’s instructions. The 16S rDNA gene was amplified using a specific primer with the barcode (16S V3 + V4). DNA sequencing libraries were generated using an NEB Next Ultra DNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA). The PCR reaction conditions consisted of 95°C for 3 min (1 cycle), 95°C for 30 s and 50°C for 30 s as well as 72°C for 30 s (25 cycles), and a final extension at 72°C for 10 min in the presence of Fast Hifidelity Polymerase and Phusion^® High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs Co., Ltd., Beijing, China). Paired-end sequencing of the PCR products was performed on a NovaSeq6000 at LC-Bio Technologies Co., Ltd. (Hangzhou, China).