DNA was extracted using the QiAmp Fast DNA Stool Mini kit (Qiagen Ltd, West Sussex, UK). Collected samples were individually placed in 2-mL tubes prefilled with 0.1 mm silica and zirconia bead mix (Benchmark Scientific, Edison, USA), dissolved in 1 mL InhibitEX buffer (Qiagen Ltd, West Sussex, UK) and vortexed until homogenized. A bead-beating step (Beadbug microcentrifuge homogenizer, Benchmark Scientific, USA) was applied for 3 x 60 s at 5 m/s with 5-min rest in between. Total genomic DNA was eluted in sterile microcentrifuge tubes and quantified by Qubit Fluorimetric Quantitation (ThermoFisher Scientific Ltd, UK) as per manufacturer’s instructions. Metataxonomic sequencing (16S rRNA gene sequencing) was performed at Research & Testing RTL Genomics (Lubbock, TX, USA), using primers detecting the V1-V2 regions of the 16S rRNA gene plus bifidobacteria regions to generate 10,000 paired-ends reads on a Illumina MiSeq (Illumina, San Diego, USA.
Qubit fluorimetric quantitation
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
The Qubit Fluorimetric Quantitation is a sensitive fluorometer designed to accurately measure the concentration of nucleic acids and proteins in sample solutions. It utilizes fluorescent dyes that selectively bind to the target molecules, providing a quantitative measurement of the sample's concentration.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Zirconia bead mix, by Benchmark Scientific (2 mentions)
Beadbug microcentrifuge homogenizer, by Benchmark Scientific (2 mentions)
Inhibitex buffer, by Qiagen (2 mentions)
Qiamp fast dna stool mini kit, by Qiagen (2 mentions)
Qiazol reagent, by Qiagen (2 mentions)
Hiscansq system, by Illumina (1 mentions)
Infinium humanmethylation450 beadchip, by Illumina (1 mentions)
Reliaprep blood gdna miniprep system kit, by Promega (1 mentions)
Ez 96 dna methylation gold kit, by Zymo Research (1 mentions)
Blood agar, by Thermo Fisher Scientific (1 mentions)
High pure polymerase chain reaction template preparation kit, by Roche (1 mentions)
Nextera xt dna sample kit, by Illumina (1 mentions)
Dneasy blood and tissue extraction kit, by Qiagen (1 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
Fastdna spin kit for soil, by MP Biomedicals (1 mentions)
Ncounter system, by NanoString (1 mentions)
8 protocols using qubit fluorimetric quantitation
1
Gut Microbiome Profiling via 16S rRNA Sequencing
2
Gut Microbiome Profiling by 16S rRNA
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A total of 29 scraped intestinal samples and 96 fecal pellets were individually placed in 2-mL tubes prefilled with 0.1 mm silica and zirconia bead mix (Benchmark Scientific, Edison, USA), dissolved in 1 mL InhibitEX buffer (Qiagen Ltd., West Sussex, UK) and vortexed until homogenized. A bead-beating step (Beadbug microcentrifuge homogenizer, Benchmark Scientific, USA) was applied for 3 × 60 s at 5 m/s with 5 min rest in-between. The DNA extraction has been performed with QiAmp Fast DNA Stool Mini kit (Qiagen Ltd., UK), following the manufacturer’s instruction. Total genomic DNA was eluted in sterile microcentrifuge tubes and quantified by Qubit Fluorimetric Quantitation (ThermoFisher Scientific Ltd., UK), following the manufacturer’s instructions. DNA aliquots were kept at − 20 °C until used. Sequencing of the variable regions of the 16S rRNA gene was performed at Research and Testing Laboratory LLC (Lubbock, Texas, USA). Primers used to amplify the V1–V2 regions of 16S rRNA gene were 28F (5′-GAGTTTGATCNTGGCTCAG-3′) and 388R (5′-TGCTGCCTCCCGTAGGAGT-3′). Sequencing was performed using an Illumina Miseq (Illumina, San Diego, USA), with 10K paired-end sequencing protocol.
3
DNA Methylation Profiling Protocol
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Buffy coats stored in liquid nitrogen were thawed, and genomic DNA was extracted using the ReliaPrep Blood gDNA Miniprep System Kit (Promega). The concentration of the genomic DNA was assessed by Qubit fluorimetric quantitation (Thermo Fisher Scientific). 500 ng of DNA was bisulfite‐converted using the EZ‐96 DNA Methylation‐Gold Kit (Zymo Research) and hybridized to Illumina Infinium HumanMethylation450 BeadChips (Illumina). Matched pairs (pre‐ and post‐intervention) were arranged randomly on the same array. All the chips were subsequently scanned using the Illumina HiScanSQ system. Control probes included in the microarray were used to assess bisulfite conversion efficiency and to exclude lower‐quality samples from further analyses.
4
Genomic DNA Extraction from L. monocytogenes
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DNA was extracted using the High Pure Polymerase Chain Reaction (PCR) Template preparation kit (Roche, Potsdam, Germany) as per manufacturer’s protocol. Briefly, pure colonies of L. monocytogenes on blood agar (Oxoid, Basingstoke, UK) were inoculated into 50 µL of DNA-free water followed by adding 200 µL of binding buffer and 40 µL of Proteinase K. The mixture was then incubated at 70 °C using heating block for 10 min. After incubation, 100 µL of isopropanol was added and the mixture was applied to a High Pure Filter tube followed by centrifugation at 13,000 rpm for a minute. The flow-through and collection tube were discarded. Then, 500 µL of inhibitor removal buffer was added on the High Pure Filter tube followed by centrifuging at 13,000 rpm for a minute and discarding of the flow-through and collection tube. The filter tube was washed two times with 500 µL of wash buffer and centrifuged at 13,000 rpm for a minute. Then, 200 µL of elution buffer (70 °C) and new collection tubes were added, followed by centrifuging at 13,000 rpm for a minute. The extracted DNA was stored at −80 °C for further analyses. The DNA quantity and purity were assessed by using Qubit fluorimetric quantitation (Thermo Fisher Scientific, Waltham, MA, USA).
5
Whole Genome Sequencing of Gram-positive Bacteria
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DNA extraction was performed by using the DNeasy Blood and Tissue Extraction Kit (QIAGEN, København Ø, Denmark), from 5 mL of liquid cultures grown overnight at 35°C in brain heart infusion medium under aerobic conditions, following the manufacturer’s protocol for gram-positive bacteria. DNA quantity and purity was assessed by using Qubit fluorimetric quantitation (Thermo Fisher Scientific, Waltham, MA, USA).
Library preparation was conducted by using the Nextera XT DNA Sample Kit (Illumina, San Diego, CA, USA). WGS was performed twice a week on a NextSeq 500 platform (Illumina) by using 2 × 150-bp runs. FqCleaner version 3.0 was used to eliminate adaptor sequences (32 (link)), reduce redundant or overrepresented reads (33 (link)), correct sequencing errors (34 (link)), merge overlapping paired reads (35 (link)), and discard reads with Phred scores (measure of the quality of identification of nucleobases generated by automated DNA sequencing)< 20. Sequences with <40 times average coverage after trimming were resequenced to avoid artifacts in allele calling (20 (link)). Assemblies were obtained by using CLC Assembly Cell version 4.3.0 (QIAGEN) with estimated library insert sizes ranging from 50 bp to 850 bp and a minimum contig size of 500 bases.
Library preparation was conducted by using the Nextera XT DNA Sample Kit (Illumina, San Diego, CA, USA). WGS was performed twice a week on a NextSeq 500 platform (Illumina) by using 2 × 150-bp runs. FqCleaner version 3.0 was used to eliminate adaptor sequences (32 (link)), reduce redundant or overrepresented reads (33 (link)), correct sequencing errors (34 (link)), merge overlapping paired reads (35 (link)), and discard reads with Phred scores (measure of the quality of identification of nucleobases generated by automated DNA sequencing)
6
Wastewater Microbiome and Resistome Profiling
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Volumes of about 100 mL from the 24 h-composite of the WWTP influent samples and about 300 mL of the WWTP effluent samples from the 24 h-composite sample were used for DNA extraction. Wastewater samples were filtered using polycarbonate membranes with a diameter of 47 mm and a pore size of 0.2 μm (Whatman Nuclepore Track-Etched Membranes, Sigma-Aldrich, Munich, Germany). DNA extraction was performed using the Fast DNA spin kit for soil (MP Biomedical, Illkrich, France) utilizing the lysing matrix E according to the manufacturer's protocol for wastewater. The quantities and purities of the DNA extracts were measured by means of the Qubit fluorimetric quantitation (Thermo Scientific, Waldham, USA).
The yield of DNA obtained from the influent water samples ranged from 71 to 244 μg per mL (n = 14) and from 15 to 143 μg per mL (n = 14) for the effluent water samples. To determine the abundances of 7 clinically relevant antibiotic resistant genes including the mcr-1 colistin resistance gene, and the 16S rRNA gene as well as the gene markers for some Enterobacteriaceae qPCR analyses were performed.
The yield of DNA obtained from the influent water samples ranged from 71 to 244 μg per mL (n = 14) and from 15 to 143 μg per mL (n = 14) for the effluent water samples. To determine the abundances of 7 clinically relevant antibiotic resistant genes including the mcr-1 colistin resistance gene, and the 16S rRNA gene as well as the gene markers for some Enterobacteriaceae qPCR analyses were performed.
7
Gene Expression Analysis of Cancer Pathways
8
DC Gene Expression Profiling
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DCs were sorted from tumors of CT26 or MC38 tumor-bearing mice via CD11c microbeads (130-125-835). The QIAZOL reagent (Qiagen Ltd) was used to extract the total RNA from DCs. The quantification of samples was assessed by Qubit Fluorimetric Quantitation (Thermo Scientific) and Nanodrop (Thermo Scientific). To analyze the gene expression profiles of six samples, the NanoString nCounter System was used by evaluating 100 ng total RNA from the sample with nCounter mouse myeloid innate immunity panel (LBL-10 398-02), which is optimized for evaluation of genes representing the overall myeloid innate immunity pathways. The correction of background was done by subtracting the geometric mean of eight negative-control probes. The values of gene expression were normalized with six positive-control probes and 32 housekeeping genes by the nSolver program, followed by log2-transformation and standardization within each sample. The fold changes and p values were displayed via volcano plots.
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