FastDigest BseLI

Protocol tips

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	The purified 16S rDNA amplicons were treated with 10 U of FastDigest BseLI (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min. An ABI PRISM 3130xl genetic analyzer (Thermo Fisher Scientific) was used to analyze the resultant DNA fragments, i.e., fluorescent-labeled terminal restriction fragments (T-RFs).

Protocol tips
The purified 16S rDNA amplicons were treated with 10 U of FastDigest BseLI (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min. An ABI PRISM 3130xl genetic analyzer (Thermo Fisher Scientific) was used to analyze the resultant DNA fragments, i.e., fluorescent-labeled terminal restriction fragments (T-RFs).

Publication protocol

The fecal samples were collected in a container with guanidine thiocyanate as a preservative solution (TechnoSuruga Laboratory, Shizuoka, Japan) and refrigerated at 4 °C until transfer to the laboratory within seven days. We conformed to the protocol [24] for the representative extraction of DNA from bacterial populations in feces. Terminal restriction fragment length polymorphism (T-RFLP) analyses to determine the relative abundance of intestinal microbiota phylogenetic groups from each fecal sample were performed at the TechnoSuruga Laboratory (Shizuoka, Japan) [25,26]. T-RFLP analysis is one of the most well-established and reliable 16S ribosomal RNA-based methods, especially when considering its high throughput and reproducibility. Briefly, the fecal samples (approximately 4 mg each) were suspended in a 1200 μL solution containing 100 mM Tris-HCl (pH 9.0), 40 mM ethylenediaminetetraacetic acid, 4 M guanidine thiocyanate, and 0.001% bromothymol blue. A FastPrep 24 device homogenized the Fecal solids in the suspension (MP Biomedicals, Irvine, CA, USA) with zirconia beads being set at 5 m/s for 2 min. DNA was then extracted from a 200 µL suspension using magLEAD 12gC (Precision System Science; Chiba, Japan). MagDEA® Dx SV (Precision System Science) was used as the reagent in automatic nucleic acid extraction. PCR was performed with a Takara Thermal Cycler Dice TP650 (Takara Bio, Shiga, Japan) in 20 µL of a reaction mixture containing 1× PCR buffer, with each deoxynucleotide triphosphate at a concentration of 200 µM, 1.5 mM MgCl2, each primer at a concentration of 0.2 µM, 10 ng of fecal DNA, and 0.2 U of HotStarTaq DNA polymerase (Qiagen, Hilden, Germany). 5′ FAM-labeled 516f (5’-TGC-CAGCAGCCGCGGTA-3’; Escherichia coli positions 516−532) and 1510r (5’-GGTTACCTTGTTACGA-CTT-3’; E. coli positions 1510−1492) were the primers used. The amplification program used was as follows: preheating at 95 °C for 15 min, 35 cycles of denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s, extension at 72 °C for 90 s, and finally, terminal extension at 72 °C for 10 min. Electrophoresis and purified using a MultiScreen PCR µ96 Filter Plate verified amplified DNA (Millipore, Billerica, MA, USA). The purified 16S rDNA amplicons were treated with 10 U of FastDigest BseLI (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min. An ABI PRISM 3130xl genetic analyzer (Thermo Fisher Scientific) was used to analyze the resultant DNA fragments, i.e., fluorescent-labeled terminal restriction fragments (T-RFs). GeneMapper software (Thermo Fisher Scientific) was used to determine the T-RF length and the peak area for each sample. T-RFs were divided into 29 operational taxonomic units (OTUs). The individual OTUs were quantified as the percentage of all OTUs combined based on the area under the curve (% AUC). The reference database, Human Fecal Microbiota T-RFLP profiling (http://www.tecsrg-lab.jp/t_rflp_hito_OTU.html), was used to putatively match the bacteria in each classification unit to the corresponding OTU. T-RFLP analyses enabled the classification of the sampled intestinal microbiota into the following 10 groups: Bifidobacterium, Lactobacillales, Bacteroides, Prevotella, Clostridium cluster IV, Clostridium subcluster XIVa, Clostridium cluster IX, Clostridium cluster XI, Clostridium cluster XVIII, and others.

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