Mp fastdna spin kit for feces

The microbial DNA of fecal samples were extracted by using the MP FastDNA Spin kit for Feces (MP Biomedicals) according to the manual. The V3-V4 region of 16S rRNA and the 60 kDa chaperonin (groEL) gene were amplified using barcoded fusion primers, and then sequenced with a Miseq TM sequencer separately for determination of microbial community and species-level Bifidobacterium composition, as previously described [29 (link)]. After sequencing, 16S rRNA sequencing data were analyzed using the QIIME pipeline [30 (link)]. For determination of bifidobacterial composition, a local nucleotide database was constructed using Bioedit, and the BLASTn algorithm was used to count the numbers of sequences that belong to each individual species. The PCA analysis based on abundances of key gut bacteria was conducted by the prcomp function and visualized using the ggbiplot package in R software. For correlation analysis, Spearman’s correlation with FDR correlation was conducted by using package psych in R. The correlation coefficients of <−0.5 or >0.5, and p < 0.05 were considered as statistically significant.

Total bacterial DNA was extracted from collected caecal samples using the MP FastDNA Spin Kit for Feces (MP Biomedicals, USA) according to the manufacturer’s instructions and quantified with the Qubit dsDNA HS Assay Kit with a Qubit 3.0 Fluorometer (ThermoFisher Scientific, USA). The average DNA concentration (± standard deviation, SD) of all animals in this study was 332 ± 134.0 ng/µL. PCR amplification of the V3–V4 regions of the 16S rRNA gene was performed using Illumina fusion primers (341F and 802R) with 2x KAPA HiFi Hot Start Ready Mix (Roche, Switzerland) with the following settings: initial denaturation at 95°C for 3 min; 25 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec; elongation at 72°C for 10 min in triplicates of each sample. Multiplexed 16S rRNA gene libraries were prepared using the Nextera XT kit (Illumina Inc., USA) followed by 2 x 250 paired-end sequencing performed with V2 chemistry on a MiSeq instrument (Illumina Inc., USA).

Microbial DNA was extracted from feces using the MP FastDNA Spin Kit for Feces (MP Biomedicals, Santa Ana, CA, USA) following the manufacturer’s instructions. The V3–4 region of 16 S rRNA gene was amplified by the primers 314 F (CCTAYGGGRBGCASCAG) and 806 R (GGACTACNNGGGTATCTAAT) jointed with a seven-base-pair barcode. The PCR product was purified by the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany), and sequenced on the Illumina Miseq platform with the Miseq Reagent Kit V3 (Illumina, San Diego, CA, USA, PE300 mode).