Total DNA was extracted from the samples of the cecal contents using a commercial DNA extraction kit (Omega Bio-Tek, Guangzhou, China) according to the manufacturer’s instructions. The DNA concentration was measured by a Qubit 3.0 (Q32866; Invitrogen, Shanghai, China). The integrity of the DNA was verified by electrophoretic analysis (FR-1000, Furi Science & Technology, Shanghai, China). DNA samples were normalized to 20 ng/μL, and the amplification of 16S ribosomal DNA V3-V4 regions was conducted using universal primers (341F: CCTACGGGNGGCWGCAG; 805R: GACTACHVGGGTATCTAATCC). The amplification program of the PCR consisted of an initial denaturation at 94 ºC for 3 min, followed by 5 cycles of 94 ºC for 30 s, 45 ºC for 20 s, and 65 ºC for 30 s, 20 cycles of 94 ºC for 20 s, 55 ºC for 20 s, and 72 ºC for 30 s, and 1 cycle of 72 ºC for 5 min, followed by holding at 10 ºC until the program was terminated. The size and concentration of the amplicons were determined by 2% agarose gel electrophoresis (FR-1000, Furi Science & Technology, Shanghai, China) and Qubit 3.0 quantitation (Q32866; Invitrogen, Shanghai, China), respectively. The amplicons were pooled in equimolar concentrations to give a final concentration of 20 pM for high-throughput sequencing.
Qubit 3
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany
The Qubit 3.0 is a fluorometer designed for accurate and sensitive quantification of DNA, RNA, and protein samples. It uses fluorescent dyes that bind specifically to the molecule of interest, providing a precise measurement of the sample concentration.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (24 mentions)
Nanodrop one, by Thermo Fisher Scientific (21 mentions)
2100 bioanalyzer, by Agilent Technologies (20 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (19 mentions)
Nanodrop, by Thermo Fisher Scientific (16 mentions)
Ampure xp bead, by Beckman Coulter (16 mentions)
Dsdna hs assay kit, by Thermo Fisher Scientific (14 mentions)
Nextseq 500, by Illumina (13 mentions)
Trizol reagent, by Thermo Fisher Scientific (13 mentions)
Qiaamp dna mini kit, by Qiagen (12 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (12 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (12 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (12 mentions)
Qiaamp circulating nucleic acid kit, by Qiagen (11 mentions)
Miseq platform, by Illumina (10 mentions)
Novaseq 6000, by Illumina (10 mentions)
Dneasy blood tissue kit, by Qiagen (10 mentions)
Dneasy blood and tissue kit, by Qiagen (10 mentions)
Tapestation 4200, by Agilent Technologies (9 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (8 mentions)
396 protocols using qubit 3
1
Cecal Microbiome DNA Extraction and Sequencing
2
SARS-CoV-2 Genome Sequencing from Nasopharyngeal Swabs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted from 300 μl of nasopharyngeal swab samples using Maelstrom 9600 (TanBead Inc., Taoyuan, Taiwan), according to the manufacturer’s instructions. PCR amplicons were prepared in accordance with the ARTIC V2 and V3 RT-PCR protocol [nCoV-2019 sequencing protocol v2 (GunIt)].2 PCR amplicon size was inspected on 2% agarose gel. DNA concentration was measured with the Qubit dsDNA High Sensitivity assay kit on Qubit 3.0 (both Thermo Fisher Scientific, Waltham, MA, USA). We prepared NGS libraries of amplicons using the Nextera XT library preparation kit (Illumina, San Diego, CA, USA), according to the vendor’s instructions. The concentrations of NGS libraries were measured using the Qubit dsDNA High Sensitivity assay on a Qubit 3.0 instrument (Thermo Fisher Scientific). The fragment sizes were analyzed using the Agilent HS DNA Kit on the Bioanalyzer 2100 (both Agilent Technologies, Santa Clara, CA, USA). Prepared samples were sequenced using the MiSeq Reagent Kit v3 (600 cycles) on the MiSeq Sequencer, the NextSeq 500/550 High Output Kit v2.5 (300 cycles) on the NextSeq 550, or NovaSeq 6000.
3
Genomic DNA Extraction and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For short-read sequencing, genomic DNA was extracted and purified using the GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich, St. Louis, MI, USA) following the manufacturer's protocols. The purity of the DNA was assessed using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA), while the concentration was determined using the Qubit 3.0 (Thermo Scientific, Wilmington, DE, USA). DNA library construction was carried out using the KAPA HyperPlus kit, following the standard protocol. Subsequently, sequencing was performed on the Illumina MiSeq platform (Illumina, San Diego, CA, USA) using the MiSeq Reagent Kit v2 (500 cycles).
For long-read sequencing, high molecular weight genomic DNA was extracted using the MagAttract HMW DNAKit (Qiagene, Venlo. NL). The purity of the extracted DNA was assessed with the NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA), while the concentration was determined using the Qubit 3.0 (Thermo Scientific, Wilmington, DE, USA). The DNA length was confirmed using the Agilent 4200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). Ligation sequencing 1D Kit (Oxford Nanopore Technologies, Oxford, UK) was used for library preparation, and sequenced on the MinION platform (Oxford Nanopore Technologies) with the R9.4.1 flowcell.
For long-read sequencing, high molecular weight genomic DNA was extracted using the MagAttract HMW DNAKit (Qiagene, Venlo. NL). The purity of the extracted DNA was assessed with the NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA), while the concentration was determined using the Qubit 3.0 (Thermo Scientific, Wilmington, DE, USA). The DNA length was confirmed using the Agilent 4200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). Ligation sequencing 1D Kit (Oxford Nanopore Technologies, Oxford, UK) was used for library preparation, and sequenced on the MinION platform (Oxford Nanopore Technologies) with the R9.4.1 flowcell.
4
Mitochondrial DNA Sequencing Pipeline
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Full length mtDNA is split into two overlapping segments, each approximately 8.5 kb in length. The primer pairs used were 5’-GCAAATCTTACCCCGCCTG-3’/5’-AATTAGGCTGTGGGTGGTTG-3’ and 5’-GCCATACTAGTCTTTGCCGC-3’/5’-GGCAGGTCAATTTCACTGG-3’. LrPCR was performed using Phanta Max Super-Fidelity DNA Polymerase (Vazyme, China) and 50 ng of total genome DNA. LrPCR reaction condition was 95 °C for 30 s, 25 cycles of 95 °C for 15 s, 60 °C for 15 s, 72 °C for 8 min with a final extension of 72 °C for 5 min. PCR products were then purified using 1% gel and FastPure Gel DNA Extraction Mini Kit (Vazyme, China), and measured using Qubit3.0 (Thermo Fisher Scientific, Massachusetts, USA). The two segments were then pooled with equal moles and tagmented using Tn5 (5 ng amplicon1, 5 ng amplicon2, 2 μL 5 × TTBL, 1 μL TTE Mix V50), incubating at 55 °C for 10 min. The tagments were retrieved using 1 × DNA Clean Beads (Beckman Coulter, Germany), and sequencing library was then constructed using TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, China). The libraries were purified using DNA Clean Beads by 0.5 × /0.3 × double size selection and the quantification was assessed by Qubit3.0 (Thermo Fisher Scientific, Massachusetts, USA). Libraries were pooled and sequenced on Illumina NovaSeq 6000.
5
Extraction and Characterization of cfDNA and gDNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
cfDNA was extracted from the human plasma samples through centrifugation at 16000 × g for 10 min at 4°C, using the QIAamp Circulating Nucleic Acid Kit (QIAGEN, Germany, Cat. No.55134); paired gDNA was extracted from the blood cells using the QIAamp DNA Mini Kit (QIAGEN, Germany, Cat. No.51304) according to the manufacturer’s instructions. Qubit 3.0 (Thermo Fisher, USA) was used to quantify the extracted cfDNA, and Agilent 2100 bioanalyzer (Agilent, USA) was used to analyze the fragment size and purity of cfDNA. Qubit 3.0 was used to quantitatively analyze the extracted gDNA, NanoDrop 8000 (Thermo Fisher, USA) was used to purify the extracted gDNA, and agar gel electrophoresis was used to analyze the integrity of the extracted gDNA. The qualified samples that DNA >10 ng and with no degradation and no pollution will be entered into the next step of the library preparation.
6
Fecal Microbiome Profiling in Pigs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fecal materials were obtained from the individual pig by rectal stimulation on days 0 (before oxytetracycline treatment), 8 (after the oxytetracycline treatment), and 21 (two weeks after the withdrawal of oxytetracycline), and stored in sterile containers at –20 °C until processed. The total DNA was extracted from the fecal samples by the QIAamp PowerFecal Kit (Qiagen, Crawley, West Sussex, UK) following the manufacturer’s instructions with some modifications recommended by Hart et al.45 (link). The final DNA were eluted in 100 μL of 10 mM Tris buffer (pH 8) after being incubated for 5 min for maximum elution efficiency. A Qubit fluorometer (Qubit 3, Invitrogen) was used to determine the total DNA concentration, and purity was assessed via the 260/280 and 260/230 absorbance ratios using a spectrophotometre (NanoDrop® ND-1000). The samples were sent for DNA sequencing to the Teagasc Food Research Centre, Ireland. Paired-end sequencing libraries were prepared from the extracted DNA using the Illumina Nextera XT Library Preparation Kit (Illumina Inc., San Diego, CA) followed by sequencing on the Illumina NextSeq 500 platform using high-output chemistry (2 × 150 bp) according to the manufacturer’s instructions.
7
Whole-Genome Sequencing of Blood and Muscle
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was extracted from whole-blood samples or muscle samples using the DNeasy Blood & Tissue Kit (QIAGEN), following manufacturer recommendations for maximizing yield and quality. Concentration was assessed by Qubit 3 (Invitrogen) and 50 ng of DNA were used as input for whole-genome sequencing (WGS). Libraries were prepared using the Nextera DNA Library Prep protocol (Illumina catalog #FC-121-1030). Briefly, DNA was added to a 10 μl reaction containing 5 μl of TD buffer and 1 μl of tagment DNA enzyme (TDE1), then incubated at 55°C for 5 minutes. Tagmentation reactions were cleaned using 2x concentration of Ampure XP beads (Beckman Coulter), then 10 μl of cleaned DNA were added to a 24 μl PCR reaction including 1x NEBNext Q5 master mix (New England Biolabs) and 0.42 μM each of indexed P5/P7 primers. Libraries were amplified using six cycles of PCR and cleaned using 0.65x concentration of Ampure XP beads (Beckman Coulter). Libraries were pooled equimolarly and sequenced on either the Illumina HiSeq X or NovaSeq 6000 platforms (2×151 bp sequencing) to a median coverage of 11.54x.
8
Environmental Sponge Microbiota Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each environmental sponge sample collected for microbiota characterization was homogenized with 50 ml of phosphate buffer containing 0.9% NaCl in a stomacher, for 7 min at 230 rpm. Fifty milliliters of the homogenate were transferred to a sterile 50 ml conical tube and centrifuged at 11,000×g and 4 °C for 20 min (Beckman Coulter, Avanti J-26 XPI) [34 (link)]. After centrifugation, supernatants were discarded and pellets were stored at − 80 °C until DNA extraction. DNA was extracted from approximately 0.25 g of each sample using DNeasy PowerSoil DNA extraction kit (QIAGEN) following manufacturer’s protocol. Approximately 0.25 g of a sterile sponge was also sampled and used as a negative control to confirm the absence of microbial DNA contaminants on the sterile sponge. DNA extracted from the sponge was processed following the same protocol as described below for other samples. The concentration of DNA in each test sample and in the control sample was determined both spectrophotometrically using NanoDrop One (Thermo Scientific) and fluorometrically using Qubit 3 (Invitrogen) and Qubit dsDNA High Sensitivity Assay Kit. DNA samples were stored at − 80 °C until further use.
9
ChIP-qPCR Analysis of JMJD6
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromatin immunoprecipitation assays were performed from three independent biological replicates using differentiated cells (24 h). Cells were crosslinked with 1% formaldehyde at room temperature for 10 min. To inactivate formaldehyde, glycine solution was added to a final concentration of 125 mM and incubated for 5 min at room temperature. Crosslinked cells were washed twice with PBS supplemented with protease inhibitor, scraped from the plate, and resuspended in 2 ml of the same solution. The cell resuspension was centrifuged for 5 min at 5,000 x g at 4°C. The PBS was removed and the pellet was processed for chromatin digestion and immunoprecipitation using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technologies). Chromatin concentration was measured using a Qubit 3 (Invitrogen) and 2 μg were incubated with anti-JMJD6 rabbit antisera [11 (link)] or rabbit IgG (2729, Cell Signaling Technologies) as a negative control. After elution and reverse crosslinking, DNA was purified using the ChIP DNA clean & concentrator kit (Zymo Research). Recovered DNA fragments were analyzed by quantitative PCR using the SYBR Green Master Mix (Applied Biosystems). Quantification of fold enrichment of precipitated DNA fraction relative to IgG was calculated as 2-Δ(Ct sample–Ct IgG). Primers for ChIP analysis are listed in Table 3 .
10
RNA Extraction and Sequencing of Mammalian Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T and A549 cells were harvested at 24 h post-transfection as described. Total RNA extraction was using miRNeasy Micro Kit (QIAGEN, German) according to the manufacture’s instruction. The concentration of RNA was measured by Qubit 3 (Invitrogen, USA), and RNA integrity was determined using Qsep1 (BiOptic, China) capillary gel electrophoresis (CGE) with an R1 RNA cartridge or Bioanalyzer 2100 system (Agilent Technologies, CA, USA) with RNA Nano 6000 Assay Kit.
Library preparation and sequencing were performed in Novogene Corporation. Ribosomal RNA was removed using Epicenter Ribo-zero rRNA Removal Kit (Epicenter, USA), and rRNA-free residue was cleaned up by ethanol precipitation. Sequencing libraries were then generated using the rRNA-depleted RNA by NEBNext® Ultra Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. The library was sequenced on an HiSeq2500 (Illumina, USA) with 150 bp paired-end reads.
Library preparation and sequencing were performed in Novogene Corporation. Ribosomal RNA was removed using Epicenter Ribo-zero rRNA Removal Kit (Epicenter, USA), and rRNA-free residue was cleaned up by ethanol precipitation. Sequencing libraries were then generated using the rRNA-depleted RNA by NEBNext® Ultra Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. The library was sequenced on an HiSeq2500 (Illumina, USA) with 150 bp paired-end reads.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!