Genomic DNA was isolated from blood using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Extracted DNA was quantified using the Quant-iT BR Assay Kit (Invitrogen, Carlsbad, CA). Genomic libraries were prepared using the SureSelectXT Methyl-Seq Target Enrichment System for Illumina Multiplexed Sequencing (Agilent Technologies, Santa Clara, CA). Briefly, genomic DNA (3 μg per sample) was randomly sheared using an ultrasonicator (Covaris Inc., Woburn, MA), after which DNA fragments were extracted. Samples were then subjected to end repair, methylated adapter ligation, hybridization to SureSelectXT Methyl-Seq Capture Library, streptavidin bead enrichment, bisulfite conversion, and PCR amplification. Sample genomic libraries were indexed and pooled for multiplexed sequencing on an Illumina HiSeq 2500 platform (Illumina, San Diego, CA) using 101 bp paired-end reads.
Quant it br assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Quant-iT BR assay kit is a fluorescence-based method for the quantitation of proteins. It allows for the measurement of protein concentrations in solution.
Automatically generated - may contain errors
Lab products found in correlation
Dneasy blood tissue kit, by Qiagen (4 mentions)
Hiseq 2500 platform, by Illumina (3 mentions)
Sureselectxt methyl seq target enrichment system for multiplexed sequencing, by Illumina (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Ultrasonicator, by Covaris (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Hiseq 4000 platform, by Illumina (1 mentions)
Quant it dsdna high sensitivity assay kit, by Thermo Fisher Scientific (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
Paired end dna sample prep kit, by Illumina (1 mentions)
Nanodrop, by Tecan (1 mentions)
Novaseq 6000 platform, by Illumina (1 mentions)
9 protocols using quant it br assay kit
1
Genomic DNA Extraction and Methylation Sequencing
2
Genome Sequencing and Variant Analysis of Rice Accessions
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Genomic DNA of 294 rice accessions was extracted using DNeasy Plant Mini Kits (QIAGEN, Hilden, Germany), according to the kit manual instructions from 0.5 g of young leaf tissues. Quality of DNA was checked by diluting a sample extracted using a Quant-iT BR assay kit (Q32850, Invitrogen) and then measuring the concentration and quality of DNA. Tecan F200 (Tecan, Männedorf, Switzerland) was used to measure the optical density value, and the extracted DNA was confirmed by electrophoresis at 1% agarose gel. Genome re-sequencing was performed on an Illumina HiSeq 2500 platform (Illumina, Inc., San Diego, CA, USA), according to its standard sequencing protocol. Data analysis was compared with IRSGP1.0 downloaded from NCBI (https://www.ncbi.nlm.nih.gov/assembly/GCF_001433935.1/ accessed on 30 July 2019), which is a standard genome sequence of rice [26 (link)]. Raw data files from short pair-end sequencing reads were first filtered using the software Cutadapt and Sickle, respectively [31 (link),32 (link)], and then mapped to the reference genome using BWA software [33 (link)]. Removal of duplicate reads and SNP calling were analyzed using GATK software [34 (link)], and high-quality SNPs were filtered based on <10% of missing data and >5% of minor allele frequency. After filtering, a total of 1,842,515 SNPs were used for GWAS analysis.
3
Neonatal Cholestasis Evaluation Protocol
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Subjects included 50 patients presenting neonatal cholestasis who had visited the Department of Pediatrics at the Kyungpook National University Children's Hospital between January 2013 and July 2016 (Table 1 ). Detailed clinical and family histories were obtained. Evaluations included laboratory examination, liver ultrasonography (USG), diisopropyl iminodiacetic acid (DISIDA) scan, percutaneous cholecystocholangiography (PCC), and liver biopsy. Genetic analysis was performed when a patient was suspected of having a syndromic disease. Two families also provided genetic material for analyses to confirm the diagnosis and were subjected to genetic counseling. Blood samples were collected and genomic DNA was extracted using a QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany). DNA quality and quantity were assessed using a Qubit Fluorometer (Invitrogen, Carlsbad, CA, USA) and a Quant-iT BR assay kit (Q32850; Invitrogen). The institutional review board of Kyungpook National University Hospital approved the protocol, and informed consent forms were obtained for genetic analysis and for utilization of the results for diagnosis and research purposes from the participants or from their legal guardian (IRB no. KNUH 2016-06-011).
4
Whole-Genome and Transcriptome Sequencing of Ruditapes philippinarum
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We collected healthy R. philippinarum samples in July, 2015 from a tidal flat farm in western sea of South Korea (36°53′21.3″N 126°22′00.6″E). We collected ∼1.5-year-old R. philippinarum samples by measuring morphometric characteristics such as shell length (32–33.5 mm) and weight (7–8 g). The collected R. philippinarum samples were dissected and frozen immediately.
The nucleic acids were extracted from two R. philippinarum adult individuals for sequencing library preparation of whole-genome and transcriptome, respectively. The dissected tissues were frozen in liquid nitrogen and homogenized. DNA was extracted from four tissues including mouth, gill, adductor muscle, and foot using Qiagen DNeasy Blood & Tissue Kit (Qiagen, Germany) according to the manufacture’s protocol. Extracted DNA was measured by Quant-iT BR assay kit (Invitrogen). Total RNAs were independently extracted from gill, adductor muscle, and foot using Trizol reagent (Invitrogen, USA). In order to increase purity and prevent genomic DNA contamination, we carried out RNA purification using RNeasy Mini Kit (Qiagen). Then, the quality of RNAs was checked by 28S/18S ratio, and RNA integrity number (RIN) value using TECAN Infinite F200 and Agilent Bioanalyzer 2100 system. All RNAs extracted from each three tissue shows 6.7–6.9 RIN values.
The nucleic acids were extracted from two R. philippinarum adult individuals for sequencing library preparation of whole-genome and transcriptome, respectively. The dissected tissues were frozen in liquid nitrogen and homogenized. DNA was extracted from four tissues including mouth, gill, adductor muscle, and foot using Qiagen DNeasy Blood & Tissue Kit (Qiagen, Germany) according to the manufacture’s protocol. Extracted DNA was measured by Quant-iT BR assay kit (Invitrogen). Total RNAs were independently extracted from gill, adductor muscle, and foot using Trizol reagent (Invitrogen, USA). In order to increase purity and prevent genomic DNA contamination, we carried out RNA purification using RNeasy Mini Kit (Qiagen). Then, the quality of RNAs was checked by 28S/18S ratio, and RNA integrity number (RIN) value using TECAN Infinite F200 and Agilent Bioanalyzer 2100 system. All RNAs extracted from each three tissue shows 6.7–6.9 RIN values.
5
Nucleic Acid Extraction and Quality Assessment
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About 3×3×3-mm3 (0.1 g) tissues were used to extract nucleic acid. Extracted DNA was quantified by Quant-iT BR assay kit (Invitrogen, Carlsbad, CA). Total RNAs from independent tissues were isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany). The 10 μL (100 ng/μL) DNA and the 10 μL (250 ng/μL) RNA of each samples were sent to proceed genetic analysis. The quality of RNAs was checked via 28S/18S ratio and RNA integrity number (RIN) value using the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA). Total RNAs with RIN values greater than 7.0 were taken for subsequent downstream analysis.
6
Genome-wide DNA Methylation Profiling
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Genomic DNA was isolated from blood using the DNeasy Blood & Tissue Kit (Qiagen, Germany) according to the manufacture’s protocol. Extracted DNA was quantified by Quant-iT BR assay kit (Invitrogen). Genomic libraries were prepared using the SureSelectXT Methyl-Seq Target Enrichment System for Illumina Multiplexed Sequencing (Agilent Technologies). Briefly, 2 μg of genomic DNA per sample were randomly sheared via ultra-sonification and DNA fragments between 150 and 200 bp were extracted. Sample DNA then underwent end repair, adapter ligation, hybridization to SureSelectXT Methyl-Seq Capture Library, streptavidin bead enrichment, bisulfite conversion, PCR amplification and were uniquely indexed using a 6-letter sequencing tag following the manufacturer’s protocol. Sample genomic libraries were then pooled and multiplexed in four separate lanes using 100 bp paired-end Illumina NovaSeq6000 S4 sequencing.
7
Whole-Genome Sequencing of C57BL/6N Mice
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Samples from the Korl, Crl, and Tac lines of the C57BL/6N strain were obtained from the National Institute of Food and Drug Safety Evaluation, Korea. Genomic DNA was extracted from the tail tissues of each mouse. Quality assessment of the DNA was performed based on the fluorescence concentration (≥50 ng/μL and total ≥3 μg) using the Quant-iT BR assay kit (Invitrogen), ultraviolet absorbance (OD260/280 ≥ 1.7 and OD260/230 ≥ 1.5) using a Tecan F-200 Nanodrop spectrophotometer, and degradation assessment on 1% agarose gels (major band of over 10 kb on the gel).
From the DNA samples that met the above quality assessment criteria, 1 μg gDNA was randomly sheared using the Covaris System. The TruSeq DNA PCR-free system (Illumina) was used for library construction following the manufacturer guidelines. Whole-genome sequencing was performed using the Illumina Hiseq. 4000 platform. In addition, we downloaded the public whole-genome sequence data of three C57BL/6N samples from the National Center for Biotechnology Information Sequence Read Archive database. The sequence quality of raw data was assessed with FastQC software13 , and Trimmomatic-0.3314 was used to eliminate potential adapter sequences and low-quality bases.
From the DNA samples that met the above quality assessment criteria, 1 μg gDNA was randomly sheared using the Covaris System. The TruSeq DNA PCR-free system (Illumina) was used for library construction following the manufacturer guidelines. Whole-genome sequencing was performed using the Illumina Hiseq. 4000 platform. In addition, we downloaded the public whole-genome sequence data of three C57BL/6N samples from the National Center for Biotechnology Information Sequence Read Archive database. The sequence quality of raw data was assessed with FastQC software13 , and Trimmomatic-0.3314 was used to eliminate potential adapter sequences and low-quality bases.
8
Genomic DNA Library Preparation for Illumina Sequencing
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Quality control (QC) for prepared genomic DNA was performed; A260/280 > 1.6 and A260/230 > 1.6 for UV absorbance using Nanodrop (Tecan) and ≥5 ug fluorescence concentration using the Quant-iT BR assay kit (Invitrogen). DNA was run on 0.7 % agarose gels to assess degradation.
Genomic DNA was fragmented into approximately 100–300-bp insert sizes by sonication. Each DNA fragment was end-repaired using the Paired-End DNA Sample Prep Kit (Illumina; San Diego, CA, USA), followed by the addition of a 3′-A overhang and ligation of the adapters. Size-selection for DNA fragments of 200–300 bp was performed using a gel extraction kit (Qiagen). The constructed DNA samples were quantified using Quant-iT™ dsDNA High Sensitivity Assay Kit (Invitrogen; Carlsbad, CA, USA) on an Agilent 2100 Bioanalyzer (Agilent Technologies; Santa Clara, CA, USA). After qPCR validation, the resulting libraries were subjected to paired-end sequencing with a 100-bp read length using the Illumina HiSeq 2500 platform (Illumina). Raw image files were processed by Illumina Real-Time Analysis (RTA) for image analysis and base calling.
Genomic DNA was fragmented into approximately 100–300-bp insert sizes by sonication. Each DNA fragment was end-repaired using the Paired-End DNA Sample Prep Kit (Illumina; San Diego, CA, USA), followed by the addition of a 3′-A overhang and ligation of the adapters. Size-selection for DNA fragments of 200–300 bp was performed using a gel extraction kit (Qiagen). The constructed DNA samples were quantified using Quant-iT™ dsDNA High Sensitivity Assay Kit (Invitrogen; Carlsbad, CA, USA) on an Agilent 2100 Bioanalyzer (Agilent Technologies; Santa Clara, CA, USA). After qPCR validation, the resulting libraries were subjected to paired-end sequencing with a 100-bp read length using the Illumina HiSeq 2500 platform (Illumina). Raw image files were processed by Illumina Real-Time Analysis (RTA) for image analysis and base calling.
9
Whole Genome Sequencing with Illumina NovaSeq 6000
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WGS was performed using the Illumina NovaSeq 6000 platform and clinical information from the KGP was matched (38, 39) . Of the 596 patient samples, 265 had already been sequenced in a previous study (40), and the remaining 331 samples were sequenced for this study (Table 1). Genomic DNA was isolated from the plate using a DNeasy Blood & Tissue kit (Qiagen, Germany) according to the manufacturer's protocol. The extracted DNA was quantified using a Quant-iT BR assay kit (Invitrogen, USA). High-molecular-weight genomic DNA was sheared using a Covaris S2 ultrasonicator system to obtain fragments of appropriate sizes.
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