All samples were processed by Shanghai Personal Biotechnology Co., Ltd (Shanghai, China), and the total microbial genomic DNA from each tube was extracted following the procedure for extracting nucleic acid instructions from the Omega Soil DNA Kit (D5625-01) kit (Omega Bio-Tek, Norcross, GA, USA). The quantity and quality of extracted DNAs were measured using a NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively. PCR amplification of the bacterial 16S rRNA genes V3–V4 region was performed using the forward primer 338F (5’-ACTCCTACGGGAGGCAGCA-3’) and the reverse primer 806R (5’-GGACTACHVGGGTWTCTAAT-3’). PCR products were quantified after purification of PCR amplicons and then mixed according to the amount of data required for each sample. Finally, the library was constructed using Illumina’s TruSeq Nano DNA LT Library Prep Kit, and 2×250bp bipartite sequencing was performed on an Illumina NovaSeq machine using the NovaSeq 6000 SP Reagent Kit (500 cycles) (Li C. et al., 2022 (link)).
Nanodrop nc2000
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NanoDrop NC2000 is a spectrophotometer designed for the measurement of DNA, RNA, and protein samples. It utilizes a small sample volume, typically 1-2 μL, to provide accurate and reproducible results. The instrument's core function is to measure the absorbance of the sample, allowing users to determine the concentration and purity of their samples.
Automatically generated - may contain errors
Lab products found in correlation
Omega soil dna kit, by Omega Bio-Tek (14 mentions)
Miseq platform, by Illumina (4 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (3 mentions)
Vahtstm dna clean beads, by Vazyme (3 mentions)
Miseq reagent kit v3, by Illumina (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Truseq nano dna lt library prep kit, by Illumina (1 mentions)
Novaseq machine, by Illumina (1 mentions)
Novaseq 6000 sp reagent kit, by Illumina (1 mentions)
Trace1300 tsq9000 triple quadrupole gc ms ms system, by Thermo Fisher Scientific (1 mentions)
E z n a stool dna kit, by Omega Bio-Tek (1 mentions)
Novaseq 6000 platform, by Illumina (1 mentions)
Omega dna kit, by Omega Bio-Tek (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
Pcr 96 flt c, by Corning (1 mentions)
Soil dna kit, by Omega Bio-Tek (1 mentions)
Miseq pe250 sequencer, by Illumina (1 mentions)
Axyprep dna gel extraction kit, by Corning (1 mentions)
Quantus fluorometer, by Promega (1 mentions)
32 protocols using nanodrop nc2000
1
Microbial Diversity Analysis via 16S rRNA
2
Microbial Community and SCFA Analysis
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Total genomic DNA samples were extracted using the OMEGA Soil DNA Kit (M5635-02) (Omega Bio-Tek, Norcross, GA, USA), following the manufacturer’s instructions, and the quantity and quality of extracted DNAs were measured using a NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). After extracting the microbial community DNA, variable regions V3–V4 of bacterial 16S rRNA gene were amplified with degenerate PCR primers, 338F (5’- ACTCCTACGGGAGGCAGCA-3’) and 806R (5’- GGACTACHVGGGTWTCTAAT-3’). Gut microbiota composition was assessed using Illumina MiSeq platform and QIIME2-based microbiota analysis in Shanghai Personal Biotechnology Co., Ltd (Shanghai, China).
The cecal contents were resuspended in a saturated NaCl solution. The samples were acidified with sulfuric acid (10%) and fatty acids were extracted with diethyl ether. The mixture was centrifuged at 12,000 rpm for 10 min and Na2SO4 was then added to the supernatant to remove the water content. The concentrations of SCFAs in the samples were analyzed by gas chromatography-mass spectrometry (GC-MS) on a TRACE1300-TSQ9000 Triple Quadrupole GC-MS/MS System (Thermo Fisher Scientific, Waltham, MA, USA).
The cecal contents were resuspended in a saturated NaCl solution. The samples were acidified with sulfuric acid (10%) and fatty acids were extracted with diethyl ether. The mixture was centrifuged at 12,000 rpm for 10 min and Na2SO4 was then added to the supernatant to remove the water content. The concentrations of SCFAs in the samples were analyzed by gas chromatography-mass spectrometry (GC-MS) on a TRACE1300-TSQ9000 Triple Quadrupole GC-MS/MS System (Thermo Fisher Scientific, Waltham, MA, USA).
3
Bat Fecal DNA Extraction and Analysis
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Fifty-three fecal samples were used, including 23 from the wild bats and 30 from the captive bats. DNA was extracted from all fecal samples using the E.Z.N.A.®Stool DNA Kit (Omega Bio-Tek, Inc., Norcross, GA, USA) per the manufacturer’s instructions and stored at −20 °C for further analysis. Extracted DNA was measured using a NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis to estimate DNA quantity and quality, respectively.
4
16S rRNA Gene Sequencing of Gut Microbiome
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The bacterial DNA from each biological sample was extracted from intestinal contents in DLC using a OMEGA DNA Kit (M5635-02) (Omega Bio-Tek, Norcross, GA, United States), following the manufacturer’s instructions. The quality of the DNA samples was assessed using a NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States) and agarose gel electrophoresis. All DNA samples were diluted to 1 ng/μL using sterile water. The V3-V4 region of 16S rRNA genes was amplified by polymerase chain reaction (PCR) using the forward primer 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and the reverse primer 806R (5′-GGACTACHVGGGTWTCTAAT-3′). PCR amplicons were purified with Vazyme VAHTSTM DNA Clean Beads (Vazyme, Nanjing, China) and quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, United States). Purified amplicons were sequenced on the Illumina NovaSeq 6,000 platform (Illumina, San Diego, United States) by Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China). The read sequences were clustered into operation taxonomy units (OTUs) with a 97% similarity cut-off using Vsearch software. The representative read of each OTU was selected using the QIIME two and R packages (v3.2.0).
5
Microbial DNA Extraction and 16S rRNA Sequencing
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The total genomic DNA samples were extracted from the samples of the gut contents, using the bacterial DNA Kit (OMEGA, Shanghai, China). The quantity and quality of extracted DNA were determined by a NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis. The forward primer 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and the reverse primer 1492R (5′-GGACTACHVGGGTWTCTAAT-3′) were used for the PCR amplification of the bacterial 16S rRNA gene. The 16S rRNA gene was amplified using a polymerase chain reaction (PCR) using a Q5 high-fidelity DNA polymerase (New England BioLabs, Beijing, China). The PCR products were detected using a 2% agarose gel electrophoresis and purified using a Axygen®AxyPrep DNA gel extraction kit. The recovered PCR amplification products were quantified through fluorescence using the Quant-it PicoGreen dsDNA Assay Kit. According to the fluorescence quantitative results, the samples were mixed in proportion to the sequencing requirements of each sample. The sequencing was completed by Paiseno Biological Co., LTD (Shanghai, China).
6
Gut Microbiome 16S rRNA Sequencing
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At the end of the experiment, the cecum of the mice was collected in a sterile environment, and the foecal contents were separated immediately into cryotubes and stored at − 80 °C. An OMEGA Soil DNA Kit (M5635-02; Omega Bio-Tek, Norcross, GA, USA) was used to extract total genomic DNA of samples. Both quality and quantity of extracted DNA were measured by NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively. It should be noted that bacterial 16S rRNA genes (V3–V4 region) were amplified using the forward primer 338F (5'-ACTCCTACGGGAGGCAGCA-3') and reverse primer 806R (5'-GGACTACHVGGGTWTCTAAT-3'). Purified and quantified PCR amplicons were sequenced using the Illumina MiSeq Platform with MiSeq Reagent Kit v3 by Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China). Where, microbiome bioinformatics analyses were conducted using QIIME2 2019.4 [23 (link)], with few modifications from the tutorial guidelines (https://docs.qiime2.org/2019.4/tutorials/ ). Sequence data analyses were performed using QIIME2 and corresponding packages in R (v3.2.0). Operational taxonomic unit (OTU) assignments of high-quality sequences (sharing ≥ 97% similarity) to clusters were performed using UCLUST.
7
Soil Nitrogen Cycling Gene Quantification
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Following the manufacturer’s instruction, soil DNA (10 cm, 30 cm, 60 cm, 100 cm, 140 cm, 200 cm) were extracted by Mag-Bind soil DNA Kit (Omega, cat: M5635-02, Radnor, PA, USA), and then we used a UV quantitative device (Nanodrop NC2000, Thermo Scientific, Waltham, MA, USA) and 1.2% agarose gel electrophoresis to determine the concentration and quality of the extracted DNA.
The absolute abundances of functional genes of the N cycle were measured by quantitative polymerase chain reaction (qPCR). The following genes were assessed: ammonia oxidation (AOA-amoA and AOB-amoA) for nitrification; nitrite reduction (nirS and nirK), nitrate reduction (narG), and nitrous oxide reduction (nosZ) for denitrification. The qPCR analyses were performed using Roche LightCycler480 Real-time PCR System with 96-well plates (Axygen, PCR-96-FLT-C, Union City, CA, USA). The primer information can be found inTable S1 . Cycle condition was 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. For all assays, the amplification efficiency ranged between 87% and 96%, and R2 values were all greater than 0.99 (Table S1 ).
The absolute abundances of functional genes of the N cycle were measured by quantitative polymerase chain reaction (qPCR). The following genes were assessed: ammonia oxidation (AOA-amoA and AOB-amoA) for nitrification; nitrite reduction (nirS and nirK), nitrate reduction (narG), and nitrous oxide reduction (nosZ) for denitrification. The qPCR analyses were performed using Roche LightCycler480 Real-time PCR System with 96-well plates (Axygen, PCR-96-FLT-C, Union City, CA, USA). The primer information can be found in
8
Soil DNA Extraction and Quantification
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Total genomic DNA samples were extracted using the OMEGA Soil DNA Kit (M5635-02) (Omega Bio-Tek, Norcross, GA, United States), following the manufacturer’s instructions, and stored at –20°C prior to further analysis. The quantity and quality of the extracted DNA were measured using a NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States) and 1.0% agarose gel electrophoresis, respectively.
9
Neonatal Fecal Microbiome DNA Extraction
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Neonatal faecal specimens were collected in sterile containers and stored at -80 °C, and microbial DNA was extracted from the faecal samples using a Soil DNA Kit (M5635-02, Omega Bio-Tek, Norcross, GA, USA). The extraction was performed with a 96-well nucleic acid extraction instrument (NanoMagBio S-96, Thermo Fisher, USA). All procedures were carried out strictly following the instructions provided with the kit. The concentration of DNA was measured using a NanoDrop NC2000 ultraviolet spectrophotometer (Thermo Fisher, USA), and the quality of the DNA was assessed via 1% agarose gel electrophoresis. The DNA concentration was adjusted, the working solution was stored at 4 °C, and the storage solution was stored at -20 °C.
10
Soil DNA Extraction and Quantification
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Total genomic DNA was extracted from each sample using the OMEGA Soil DNA Kit (M5635-02) (Omega Bio-Tek, Norcross, GA, USA), then stored at −20°C before analysis. The quantity and quality of the extracted DNA were measured using a NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively.
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