The murine fecal samples were collected from the cecum after a 4-week treatment with PHGG, placed in tubes, and weighed. Bacterial DNA was extracted using the NucleoSpin DNA Stool kit (TaKaRa Bio Inc., Shiga, Japan) according to the manufacturer’s protocol. The 16S ribosomal RNA gene sequencing was performed by Bioengineering Lab Co., Ltd. (Kanagawa, Japan). Briefly, the bacterial 16S ribosomal RNA gene amplicon sequence library was prepared. To analyze the V4 region, the first PCR was performed with Bakt_341F and Bakt_805R primers, and the second amplification was conducted with the index primers. Equimolar amounts of purified DNA amplicons were further amplified on a GridlON X5 platform (Oxford nanopore Technologies, Oxford, UK) for paired-end sequencing (2 × 300 bp) according to the standard protocols presented by Bioengineering Lab Co., Ltd. (Kanagawa, Japan). QIIME version 2.0 (2019.4) was used with the default parameter values for sequence denoising and with the DADA2 method for chimera checking. The Operational Taxonomic Units (OTUs) table was clustered with a 97% similarity cutoff based on the open-reference approach using UCLUST.
Nucleospin dna stool kit
Manufactured by Takara Bio
Sourced in Japan
The NucleoSpin DNA Stool kit is a laboratory equipment designed for the isolation and purification of genomic DNA from stool samples. It utilizes a silica-based membrane technology to efficiently capture and separate DNA from various sample components.
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5 protocols using nucleospin dna stool kit
1
Murine Gut Microbiome Profiling via 16S rRNA Sequencing
2
Murine Gut Microbiome Profiling via 16S rRNA Sequencing
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The murine fecal samples were collected from the cecum after a 4-week treatment with PHGG, placed in tubes, and weighed. Bacterial DNA was extracted using the NucleoSpin DNA Stool kit (TaKaRa Bio Inc., Shiga, Japan) according to the manufacturer’s protocol. The 16S ribosomal RNA gene sequencing was performed by Bioengineering Lab Co., Ltd. (Kanagawa, Japan). Briefly, the bacterial 16S ribosomal RNA gene amplicon sequence library was prepared. To analyze the V4 region, the first PCR was performed with Bakt_341F and Bakt_805R primers, and the second amplification was conducted with the index primers. Equimolar amounts of purified DNA amplicons were further amplified on a GridlON X5 platform (Oxford nanopore Technologies, Oxford, UK) for paired-end sequencing (2 × 300 bp) according to the standard protocols presented by Bioengineering Lab Co., Ltd. (Kanagawa, Japan). QIIME version 2.0 (2019.4) was used with the default parameter values for sequence denoising and with the DADA2 method for chimera checking. The Operational Taxonomic Units (OTUs) table was clustered with a 97% similarity cutoff based on the open-reference approach using UCLUST.
3
Fecal DNA Extraction for Microbiome Analysis
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Feces were obtained from participants and stored at − 80 °C until extraction. DNA was extracted from the fecal samples using NucleoSpin DNA Stool Kit (Takara Bio Inc., Shiga, Japan). 180–220 mg of defrosted samples was weighed in bead tubes, 850 µl first lysis buffer was added, and the suspension was shaken horizontally for a few seconds. Next, it was heated at 70 °C for 5 min, followed by a vortex on Vortex-Genie 2 (Scientific Industries, United States) at max speed for 10 min. The suspension was centrifuged at 13,000 × g for 3 min and 100 µl second lysis buffer was added to 600 µl of supernatant. The lysate was vortexed for 5 s and incubated at 4 °C for 5 min. After centrifugation at 13,000 × g for 3 min, the cleared lysate was obtained. The remaining steps, including wash and extraction, were performed according to the manufacturer’s protocol. Finally, DNAs were suspended in a 30 µl extraction buffer and stored immediately at -80 °C.
4
Gut Microbiome Modulation via Ang4 Administration
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All sequencing procedures were performed on Illumina MiSeq platforms with primers targeting the variable regions V3 and V4 of the 16S rRNA gene. Briefly, 50 μg of Ang4 in 100 μL PBS were administered rectally to 7-week-old female C57BL/6J mice on days 0, 2, and 4. The same volume of PBS was used for each mouse as a control. After three administrations of Ang4 and PBS, feces were collected on the subsequent day and stored at −80°C until DNA extraction. DNA was isolated from frozen stool samples using the NucleoSpin DNA Stool kit (Takara) in accordance with the manufacturer’s instructions. The V3–V4 regions of the 16S rRNA gene were amplified by PCR and sequenced on a MiSeq platform (Illumina). The raw reads were processed and analyzed using the QIIME 2 software package.1 DADA2 was used for forward and reverse sequence merging, quality checking, and amplicon sequence variants (ASV) clustering. Silva 132 sequencing database was used for taxonomic classification. Furthermore, alpha diversity (Shannon entropy) and beta diversity (unweighted UniFrac measures) were calculated to determine the overall microbial community differences between the two groups using the same software packages. To examine the significant differences in bacterial taxa (phyla, classes, families, and genera), the relative abundance was measured for each sample.
5
Quantifying Gut Microbiome Changes Using qPCR
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Fecal DNA was extracted from mouse stool samples by the NucleoSpin DNA Stool kit (Takara Bio, Kusatsu, Japan) as per the manufacturer's instructions. Six primer sets (Table 1) were prepared to analyze the changes in the abundance of intesti- https://doi.org/10.14407/jrpr.2020.45.4.163 JRPR nal flora by real-time polymerase chain reaction (PCR) [16, 17] with the Power SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) and the Step One Plus instrument (Applied Biosystems, Waltham, MA, USA). Each cycle threshold (CT) value of the targeted bacteria was corrected by the CT value for the "all bacteria" PCR product, and compared with the respective CT value before irradiation to determine the relative abundance (ΔΔCT method). The PCR conditions were 40 cycles of thermal denaturation at 95°C for 15 seconds, annealing for 30 seconds, and elongation at 80°C for 30 seconds. The annealing temperature was set for each primer as indicated in Table 1. CT values of all fecal DNA samples were measured thrice with triplicates. Relative bacteria frequency is indicated as mean ± standard deviation.
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