Fecal samples were collected after all behavioral tests (Figure 1 A). These were placed in 1.5‐mL tubes, snap frozen on dry ice, and stored at −80°C prior to 16S rRNA analysis of the samples at Oe Biotech Co., Ltd., Shanghai, China. DNA extraction was performed using TIANamp Stool DNA Kits (Tiangen Biotechnology Company, Beijing, China). Genomic DNA was then amplified in 50‐μL triplicate reactions with primers specific to the V3‐V4 region of the bacterial 16S rRNA gene: 338F (5′‐ACTCCTACGGGAGGCAGC‐3′) and 806R (5′‐GG ACTACHVGGGTWTCTAAT‐3′). The reverse primer contained a sample barcode, and both primers were connected with an Illumina sequencing adaptor (Illumina company, San Digeo, CA, USA). The PCR products were purified, and the concentrations were adjusted for sequencing on an Illumina Miseq PE300 system. The original sequencing reads from the samples were sorted by the unique barcodes, and the barcodes, linkers, and PCR primer sequences were then removed. The resultant sequences were screened for quality, and 70 or more base pairs were selected for the bioinformatics analysis. All the sequences were classified using the NCBI BLAST and SILVA databases. Distance calculations, operational taxonomic unit clustering, rarefaction analysis, and estimator calculation (for α‐diversity and β‐diversity) were performed with the MOTHUR program.24
Tianamp stool dna kit
Manufactured by Tiangen Biotech
Sourced in China
The TIANamp Stool DNA Kit is a product designed for the extraction and purification of DNA from stool samples. It is a laboratory equipment used to isolate and concentrate DNA from complex stool matrices.
Automatically generated - may contain errors
Lab products found in correlation
Miseq platform, by Illumina (22 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (12 mentions)
Nanodrop, by Thermo Fisher Scientific (10 mentions)
Axyprep dna gel extraction kit, by Corning (7 mentions)
Quantifluor st, by Promega (7 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (7 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (7 mentions)
Novaseq platform, by Illumina (7 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (5 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (5 mentions)
Hiseq 2500, by Illumina (5 mentions)
Hiseq 2500 platform, by Illumina (5 mentions)
Novaseq 6000, by Illumina (5 mentions)
Nanodrop 2000 uv vis spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (4 mentions)
Ampure xp bead, by Beckman Coulter (4 mentions)
Miseq system, by Illumina (4 mentions)
Novaseq 6000 platform, by Illumina (4 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (3 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (3 mentions)
238 protocols using tianamp stool dna kit
1
Fecal Microbiome Analysis Using 16S rRNA Sequencing
2
Gut Microbiome Analysis via 16S rRNA
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Fresh stool sample were collected in month 8 and immediately stored at −80 °C for subsequent analysis. The sample sizes were 5 in control group,5 in HFD group, and 4 in HFD + BBR group. There is no difference in sampling between the groups studied. Genomic DNA of microbiota was extracted from fecal sample by TIANamp stool DNA kits (TIANGEN). DNA was quantified by the Nanodrop 2000. The extracted DNA from each sample was used as the template to amplify the V3 and V4 hypervariable regions of ribosomal 16S rRNA genes. Briefly, the purified 1 μg of genomic DNA were fragmented to an average size of 300-400 bp and ligated with adapters. The PCR was performed using a primer cocktail that anneals to the ends of the adapters to enrich DNA fragments that have adapter molecules on both ends and followed by clean up and quantification. Sequencing was performed using a 300-bp paired-end sequencing protocol on the Illumina MiSeq platform (Illumina, San Diego, CA, USA) at Oebiotech Company, Shanghai, China. Raw paired-end reads were subjected to quality filtering using Trimmomatic software before paired-end read assembling with FLASH software. All chimeras of assembling sequences were eliminated to reach high-quality sequences.
3
Illumina-based 16S rRNA sequencing of gut microbiome
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DNA extraction was performed using TIANamp stool DNA kits (Tiangen Biotechnology Company, Beijing, China). For the Illumina MiSeq sequencing, the PCR amplification of the V3-V4 region of the bacterial 16S rRNA was performed using the following primers: 338F (5′-ACTCCTACGGGAGGCAGC-3′) and 806R (5′-GG ACTACHVGGGTWTCTAAT-3′). Sequencing was conducted using a paired-end 2 × 300 base pairs (bp) cycle run on an Illumina MiSeq platform (Berry Genomics Co., Ltd, Beijing, China). Reads obtained from the samples were sorted by unique barcodes of each PCR product. The barcode, linker, and PCR primer sequences were removed from the the original sequencing reads. The resultant sequences were screened for quality, and only sequences with ≥70 bp were selected for bioinformatics analysis. NCBI BLAST and SILVA databases were used to classify all sequences. Distance calculation, operational taxonomic units cluster, rarefaction analysis and α-diversity were performed by the MOTHUR program.
4
Gut Microbiome Sequencing from Mouse Colon
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Bacteria genomic DNA was extracted from the mice colon contents with TIANamp Stool DNA Kits (TIANGEN BIOTECH, cat. #DP328-02, Beijing, China) according to the manufacturer’s instructions. DNA concentration and purification were measured with a Nanodrop 2000 UV–vis spectrophotometer (Thermo Scientific, Wilmington, USA) and DNA quality was checked by 1% agarose gel electrophoresis. The V3-V4 hypervariable regions of 16S ribosomal RNA genes were amplified using barcoded primers 338 F and 806 R by thermocycler PCR system (GeneAmp 9700, ABI, USA). The PCR amplicons were purified by AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), and quantified by QuantiFluor™ -ST (Promega, USA). Purified amplicons were sequenced on an Illumina MiSeq platform (Illumina, San Diego, USA) according to the standard protocols by Majorbio BioPharm Technology Co. Ltd. (Shanghai, China).
5
Intestinal Microbiome DNA Extraction
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Total DNA was extracted from the intestinal contents using TIANamp Stool DNA Kits (Tiangen), following the manufacturer's instructions. A NanoDrop 2000 (Thermo Scientific) and agarose gel electrophoresis were used to determine DNA quantity and quality.
6
Fecal Microbiome Analysis: 16S rRNA Sequencing
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The fecal samples were collected immediately after behavioral tests (Fig. 1A and Fig. 4A ). Samples were placed in 1.5 ml tubes, snap-frozen on dry ice, and stored at −80°C. The 16S rRNA analysis of the fecal samples was performed by OE Biotech Co., Ltd. (Shanghai, China). DNA extraction was performed using TIANamp stool DNA kits (Tiangen Biotechnology Company, Beijing, China). Genomic DNA was amplified in 50 μL triplicate reactions with bacterial 16S rRNA gene (V3–V5 region)-specific primers: 338F (5′-ACTCCTACGGGAGGCAGC-3′) and 806R (5′-GG ACTACHVGGGTWTCTAAT-3′). The reverse primer contained a sample barcode and both primers were connected with an Illumina sequencing adapter. PCR products were purified and the concentrations were adjusted for sequencing on an Illumina Miseq PE300 system. Original sequencing reads from the sample were sorted by unique barcodes, followed by the removal of the barcode, linker, and PCR primer sequences. The resultant sequences were screened for quality, and ≥70 base pairs were selected for bioinformatics analysis. All sequences were classified using the NCBI BLAST and SILVA databases. Distance calculation, operational taxonomic units cluster, rarefaction analysis, and estimator calculation (α-diversity and β-diversity) were performed by the MOTHUR program [37 (link)].
7
Molecular Detection of Giardia and Cryptosporidium in Environmental Samples
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DNA was extracted from human and animal stool samples using TIANamp Stool DNA kits (Tiangen Biotech, Beijing, Cat. No. DP328) and from fly and water samples using TIANamp Genomic DNA kits (Tiangen Biotech, Beijing, Cat. No. DP304) following the manufacturer’s instructions. Extracted DNA was then stored at -80° C until PCR amplification.
Target genes comprised the 18S ribosomal RNA (rRNA) gene for Giardia duodenalis and the Cryptosporidium Oocyst Wall Protein (COWP) for Cryptosporidium spp. [70 (link),71 (link)]. Modified amplification reactions were performed with optimized concentrations for primer and probe sequences described in previous studies [70 (link),71 (link)]. Amplification consisted of three minutes at 95°C followed by 40 cycles of 30 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C then finally 7 minutes at 72°C. Amplification, detection, and data analysis were performed on the iCycler iQ Real-Time Detection System (Bio-Rad, California, USA).
Target genes comprised the 18S ribosomal RNA (rRNA) gene for Giardia duodenalis and the Cryptosporidium Oocyst Wall Protein (COWP) for Cryptosporidium spp. [70 (link),71 (link)]. Modified amplification reactions were performed with optimized concentrations for primer and probe sequences described in previous studies [70 (link),71 (link)]. Amplification consisted of three minutes at 95°C followed by 40 cycles of 30 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C then finally 7 minutes at 72°C. Amplification, detection, and data analysis were performed on the iCycler iQ Real-Time Detection System (Bio-Rad, California, USA).
8
DNA Extraction from Chicken Cecal Digesta
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DNA was extracted from samples of cecal digesta from 6 chickens in each treatment. The method was slightly modified from the instructions in TIANamp Stool DNA Kits from Tiangen Biotech (Beijing, PR China), as described below. The sample vortexing step was replaced with a bead-beating homogenization using 1.4-mm Ceramic Bead Tubes in a PowerLyzer-24 homogenizer (MP Biomedicals, Santa Ana, CA 92707) to enhance the cell lysis. The DNA concentrations of the extracts were measured fluorometrically with the Qubit dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA), after which the DNAs were stored at −80°C until 16S rDNA library preparation.
9
Broiler Gut Microbiome Profiling
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Genomic DNA was extracted from cecal contents of 6 broilers in each treatment using TIANamp Stool DNA Kits from Tiangen Biotech (Beijing, China). PCR amplification and sequencing were performed by the G-BIO Inc. (Hangzhou, China). The V3 and V4 regions were amplified using forward primers containing the sequence 5′-CCTACGGGNGGCWGCAG-3′ and reverse primers containing the sequence 5′-GACTACHVGGGTATCTAATCC-3′.
The processed pair-end reads were assembled using PandaSeq v2.8 with default parameters. Chimeras were identified and removed using USEARCH 6.1 within QIIME. The QIIME script “add_qiime_labels.py” was used to combine the non-chimeric sequences from each sample into 1 file. OTU picking and taxonomic assignments was performed using the open-reference OTU picking workflow in Qiime with the Greengenes reference database August 2013 release (Lee et al., 2012 (link)). OTUs with abundance below 0.005% of the total number of sequences were discarded (Bokulich et al., 2013 (link)). Alpha diversity measurements, including Shannon (Chao and Shen, 2003 ), Chao1 (Chao, 1984 ), observed species, and Good's coverage (Good, 1953 ), were calculated using the alpha_rarefaction.py script in QIIME. Weighted and unweighted unifrac distances (Lozupone and Knight, 2005 (link)) were calculated from the rarefied OTU table using the beta_diversity_through_plots.py script in QIIME.3.2.5.1.
The processed pair-end reads were assembled using PandaSeq v2.8 with default parameters. Chimeras were identified and removed using USEARCH 6.1 within QIIME. The QIIME script “add_qiime_labels.py” was used to combine the non-chimeric sequences from each sample into 1 file. OTU picking and taxonomic assignments was performed using the open-reference OTU picking workflow in Qiime with the Greengenes reference database August 2013 release (Lee et al., 2012 (link)). OTUs with abundance below 0.005% of the total number of sequences were discarded (Bokulich et al., 2013 (link)). Alpha diversity measurements, including Shannon (Chao and Shen, 2003 ), Chao1 (Chao, 1984 ), observed species, and Good's coverage (Good, 1953 ), were calculated using the alpha_rarefaction.py script in QIIME. Weighted and unweighted unifrac distances (Lozupone and Knight, 2005 (link)) were calculated from the rarefied OTU table using the beta_diversity_through_plots.py script in QIIME.3.2.5.1.
10
Intestinal Microbiome Extraction and Analysis
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The intestinal contents from the three fish were mixed, transferred to a sterile freezing tube, snap-frozen in liquid nitrogen, and stored at −80°C (Haier, DW-86L626, China) for DNA extraction. Total DNA was extracted from the intestinal contents using TIANamp Stool DNA Kits (Tiangen), following the manufacturer’s instructions. A ultramicro biochemical spectrophotometer (Thermo Scientific, NanoDrop, 2000, China) and agarose gel electrophoresis (Beijing Liuyi Instrument Factory, DYY-6C, China) were used to determine DNA quantity and quality.
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