For BoPyV whole-genome sequencing, conducted at UdelaR, viral genomic DNA was purified from the whole genomic DNA from kidney by extracting the 4–6 kb region of a 1% agarose gel. DNA purity, integrity, and concentration were assessed with a Qubit device (Thermo Fisher Scientific). The sequencing library was prepared with the ligation sequencing kit (SQK-LSK109) following the manufacturer’s instructions and directly sequenced on a FLO-MIN106 flow cell in a MinION device (Oxford Nanopore Technologies®, Oxford, UK) for 24 h. High-accuracy basecalling was performed with Guppy v3.6.0, and reads were trimmed and filtered with Nanofilt and Nanoplot [37 (link)]. Reads with quality over 10 were used in further analyses. A host filtering step was executed by mapping clean reads to the Bos taurus reference genome (GCF_002263795.1_ARS-UCD1.2_genomic.fna) using Minimap2 [38 (link)]. Unmapped reads were then mapped against the BoPyV-1 reference genome (NC_001442) and the consensus sequence was obtained using SAMtools [39 (link)]. The obtained complete genome sequence was deposited in GenBank. Genome annotation was performed using the BoPyV-1 reference genome (NC_001442).
Qubit device
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Qubit device is a fluorometer designed for precise quantitation of nucleic acids and proteins. It accurately measures concentrations of DNA, RNA, and protein samples using fluorescent dyes. The Qubit device provides reliable and sensitive measurements with a simple workflow.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Fragment analyzer, by Agilent Technologies (2 mentions)
Quantseq 3 mrna seq library prep kit fwd, by Lexogen (2 mentions)
Quantifluor one dsdna system, by Promega (2 mentions)
Quantus fluorometer, by Promega (2 mentions)
Minion device, by Oxford Nanopore (1 mentions)
Repli g mini kit, by Qiagen (1 mentions)
Precellys evolution, by Bertin Technologies (1 mentions)
Zymobiomics 96 magbead dna kit, by Zymo Research (1 mentions)
Qubit dna br kit, by Thermo Fisher Scientific (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Tapestation system, by Agilent Technologies (1 mentions)
Nextseq 550 system, by Illumina (1 mentions)
Total rna standard sensitivity assay, by Agilent Technologies (1 mentions)
Blackprep ffpe dna kit, by Analytik Jena (1 mentions)
Innuprep ffpe total rna kit, by Analytik Jena (1 mentions)
Maxwell rsc 48, by Promega (1 mentions)
Lookout dna erase, by Merck Group (1 mentions)
Nextseq 500 550 high output kit v2, by Illumina (1 mentions)
Bioanalyzer rna pico chip, by Agilent Technologies (1 mentions)
16 protocols using qubit device
1
Whole-Genome Sequencing of Bovine Polyomavirus
2
Synthetic Peptides for Chagas Disease Antibody Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two T. cruzi peptides, (Tc/NCT-1 = SLQDIIRGLSIPDT and Tc/NCT-4 = KSLRIPHGD W GATW), were chosen to be synthesized by the F-moc strategy in a synthesizer machine (PSS-8, Shimadzu, Kyoto, Japan) with a C-terminal cysteine that was used to conjugate peptides to bovine serum albumin (BSA) using a maleimide activated kit according to manufacturer’s instructions. The reaction mixture was passed through a centricon-P10, and the peptide concentration in the filtrate (uncoupled peptide) was measured on a Qubit device (Thermo Fisher, Waltham, MA, USA). After the coupling reaction, two New Zealand rabbits were immunized by subcutaneous injection of peptide-BSA (150 µg) emulsified with an equal volume of saponine. Three other inoculations, without adjuvant, were each administered 7 days later, and the serum was collected five days after the last injection. Blood was collected under standard bioethics conditions from the marginal ear vein.
3
Whole-Genome Amplification from Limited DNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA concentrations were determined by analyzing 1 μL per sample on a Qubit device (Thermo Fisher Scientific) according to the manufacturer’s instructions. For some samples, the available sample volume was low. We used 50.5–1270 ng gDNA for whole-genome amplification (WGA) with the REPLI-g Mini kit (Qiagen) according to the manufacturer’s instructions. We used 2.5 μL template DNA as input, incubated for 16 h at 30°C and denatured at 65°C for 3 min. A list of all samples, the available volume, DNA concentration, and details on WGA are shown in Table S1 .
4
Fecal Sampling and DNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fecal samples were collected at baseline, at 24 weeks, and at 48 weeks. Participants were provided stool collection kits and instructed to collect an at-home sample within 48 h of their next research visit. The fecal sample was collected using a collection vial and then placed immediately in the home freezer (−20 °C) before being brought to the clinic in a provided cooler bag with a cooler block. Samples received at the research center were immediately placed in a freezer at −80 °C.
Genomic DNA was extracted using the ZymoBIOMICS™ 96 MagBead DNA kit (Zymo Research Corp., Irvine, CA, USA) integrating a double lysis (mechanical and chemical) on the Precellys Evolution homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France). DNA extraction was performed on the KingFisher Flex automaton (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Once obtained, the DNA solutions were assayed by fluorimetry with the Qubit device (Thermo Fisher Scientific, Waltham, MA, USA).
Genomic DNA was extracted using the ZymoBIOMICS™ 96 MagBead DNA kit (Zymo Research Corp., Irvine, CA, USA) integrating a double lysis (mechanical and chemical) on the Precellys Evolution homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France). DNA extraction was performed on the KingFisher Flex automaton (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Once obtained, the DNA solutions were assayed by fluorimetry with the Qubit device (Thermo Fisher Scientific, Waltham, MA, USA).
5
Twist Multiplex Library Preparation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We prepared 8-plex and 96-plex multiplex libraries according to the Twist 96-Plex Library Preparation Kit protocol (Twist Bioscience) with the following modifications. For each sample, we denatured 100 ng of genomic DNA (25 ng/µL) at 98°C for 1 min. Ultralow (30%) GC random primer set A was used for the extension and termination reaction (Reaction A) followed by 8 and 9 cycles of PCR amplification for the 96-plex and 8-plex libraries, respectively. Final libraries were purified using Twist DNA Purification Beads (0.65× volume) and a second round of purification was applied to the supernatant using 10 µL of beads to achieve a median insert size of 500 bp. Libraries were quantified using the Qubit BR DNA kit and a Qubit device (Thermo Fisher Scientific) and size distributions were assessed using a Tape Station System (Agilent Technologies). Nondepleted libraries were pooled at equimolar concentrations and sequenced on a NovaSeq 6000 instrument (Illumina) to generate 150-bp paired-end reads.
6
3'-mRNA Sequencing for Transcriptome Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA samples used for 3′-mRNA Seq analyses were quantified by fluorometric measurement using the Qubit device and a RNA High Sensitivity assay (Thermo Fisher Scientific Inc. Massachusetts, USA) and quality measured by capillary electrophoresis using the Fragment Analyzer and the “Total RNA Standard Sensitivity Assay” (Agilent Technologies Inc. Santa Clara, USA). All samples in this study showed high quality in RNA Quality Numbers (RQN 10). 100 ng total RNA per sample were used for library preparation performed according to the manufacturer’s protocol using the QuantSeq 3′-mRNA-Seq Library Prep Kit FWD (Lexogen®, Vienna, Austria). Bead purified libraries were normalized and finally sequenced on the NextSeq550 system (Illumina Inc. San Diego, USA), using single-end sequencing with a read length of 76bp. The bcl2fastq2 tool was used to convert the bcl files to fastq files as well as for adapter trimming and demultiplexing.
7
Genetic Diversity of Karachai Goats
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 269 Karachai goats from six breeding herds, including Darinsk, Kyzyl Kala, Maysky, Piatigorsky, Storozhevaya, and Uchkulan, were randomly selected for our study. The ear tissue samples were collected by trained personnel under strict veterinary rules in accordance with the rules for conducting of laboratory research (tests) in the implementation of the veterinary control (supervision) approved by Council Decision Eurasian Economic Commission No 80 (10 November 2017). The DNA was extracted using the DNA-Extron reagent kit (JSC Sintol, Russia) according to the manufacturer’s protocol. The concentration of double-stranded DNA was determined on a Qubit device (Thermofisher, Waltham, MA, USA). The purity of DNA was estimated by the degree of absorption at a wavelength of 260 and 280 nm, using a NanoDrop 8000 micro-spectrophotometer (Thermo Fisher Scientific, DE). The DNA samples were genotyped using the Illumina Goat SNP BeadChip (Illumina Inc., San Diego, CA, USA) comprising of 53,347 SNPs.
8
Tumor cell enrichment from FFPE tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumor cells were enriched from formalin-fixed paraffin-embedded (FFPE) tissue slides from biopsy or surgical specimens by microdissection. For the VP and ulcWGS assays, DNA was extracted with the Maxwell RSC 48 (Promega, Walldorf, Germany), using the Maxwell RSC FFPE Plus DNA Kit or the blackPREP FFPE DNA Kit (Analytik Jena, Jena, Germany), respectively. RNA for the FP assay was isolated by applying the innuPREP FFPE total RNA Kit (Analytik Jena). Nucleic acids were quantified by using a Quantus Fluorometer with the QuantiFluor ONE dsDNA System (Promega) or a Qubit device (Thermo Fisher Scientific, Waltham, MA, USA) with dsDNA HS or RNA HS assays, as appropriate.
9
Microbiome Profiling of Colorectal Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Stool samples were collected and stabilized before surgery and bowel preparation (stool preOp) and after surgery (stool postOp) on days 5–7 using the Omnigene Gut system (DNA Genotek, Ottawa, ON, Canada) and stored at −80 °C until DNA extraction. DNA was extracted from stool using the PSP Stool DNA stool kit according to the specifications of the manufacturer (Invitek Molecular, Berlin, Germany). Specimens of tumor tissue and mucosal tissue were collected immediately after resection, suspended in Qiagen RNA later buffer and stored at −80 °C. DNA from tumor tissue and mucosal tissue of the proximal and distal resection margins was extracted using Dulbecco’s phosphate buffered saline (Sigma Aldrich Chemistry GmbH, St. Louis, MO, USA) and the Qiamp Microbiome Kit (Qiagen, Hilden Germany) according to the manufacturer’s recommendations. DNA from stool samples was extracted using a PSP® Spin Stool DNA Kit (Invitek Molecular) and LookOut® DNA Erase (Sigma Life Science, St. Louis, MO, USA). DNA was subsequently quantified using a Qubit device (Thermo Fisher Scientific, Waltham, MA, USA).
10
Synthesis and Conjugation of T. cruzi Peptides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two T. cruzi peptides, EP8 and EP9 (Table 1), were chosen to be synthesized by the F-moc strategy in a synthesizer machine (PSS-8, Schimadzu, Kyoto, Japan) with a C-terminal cysteine that was used to conjugate peptides to bovine serum albumin (BSA) using a maleimide activated kit according to manufacturer’s instructions. The reaction mixture was passed through a centricon-P10 and the peptide concentration in the filtrate (uncoupled peptide) measured on a Qubit device (Thermo Fisher, Waltham, MA, USA). Efficiency was calculated as (total peptide-uncoupled peptide/total peptide), which ranged between 80% and 85% in every case. Peptides were also conjugated to biotin in the C-terminal region, as previously described [36 (link)].
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!