The quality and quantity of the isolated DNA samples were assessed by agarose gel electrophoresis and measured fluorimetrically using the Qubit® HS DNA assay (Life Technologies, Darmstadt, Germany) in institute A. The quantity of the isolated DNA was measured spectrophotometrically using the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) in institute B. In institute C, the DNA content was measured fluorimetrically using the Qubit HS DNA assay (Life Technologies) and using a qPCR-based method (RNaseP Detection system; Life Technologies).
Qubit dna hs assay
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Qubit DNA HS Assay is a sensitive fluorometric-based method for quantifying DNA concentrations. The assay uses a fluorescent dye that binds specifically to double-stranded DNA, allowing for accurate measurement of DNA samples with concentrations as low as 0.2 ng/mL.
Automatically generated - may contain errors
Lab products found in correlation
Maxwell rsc ffpe plus dna purification kit, by Promega (5 mentions)
Qubit rna hs assay, by Thermo Fisher Scientific (4 mentions)
Nextseq 500, by Illumina (4 mentions)
Rnasep detection system, by Thermo Fisher Scientific (4 mentions)
Maxwell 16 research system, by Promega (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Nextseq 500 platform, by Illumina (3 mentions)
Qiaamp dna mini kit, by Qiagen (3 mentions)
Miseq platform, by Illumina (3 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Kapa library quantification kit, by Roche (2 mentions)
Rnaseout, by Thermo Fisher Scientific (2 mentions)
Kapa hifi hotstart readymix pcr kit, by Roche (2 mentions)
Mgcl2, by Merck Group (2 mentions)
Superscript 4 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Betaine, by Merck Group (2 mentions)
Ampure xp magnetic beads, by Beckman Coulter (2 mentions)
Monarch hmw dna extraction kit for cells blood, by New England Biolabs (2 mentions)
Femto pulse system, by Agilent Technologies (2 mentions)
Qubit fluorometer, by Oxford Nanopore Technologies (2 mentions)
60 protocols using qubit dna hs assay
1
Comprehensive DNA Quantification Across Institutes
2
Exome Sequencing from Blood DNA
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DNA from blood was submitted to The Center for Applied Genomics (TCAG), Toronto, Canada for exome library preparation and sequencing. DNA was quantified by Qubit DNA HS assay (Life Technologies, Carlsbad, CA) and 100 ng of input DNA was used for library preparation using the Ion AmpliSeq Exome Kit (Life Technologies) according to the manufacturer's recommendations. The Ampliseq Exome library was immobilized on Ion PI™ Ion Sphere™ particles using the Ion PI Template OT2 200 Kit v3. Sequencing was performed with the Ion PI Sequencing 200 Kit v3 and Ion PI Chip v2 in the Ion Proton™ semiconductor sequencing system following the manufacturer's recommendation. Alignment and variant calling were performed using Torrent Suite (v4.0) on the Ion Proton Server, using the Ion Proton AmpliSeq germline low stringency setting and the hg19 reference genome. The variants were annotated using an in-house annotation pipeline (22 (link)) based on Annovar (November 2014 version) (23 (link)) and RefSeq gene models (downloaded from UCSC 01 August 2015).
3
RNA Extraction and Sequencing in Zebrafish
4
Next-Generation Sequencing Library Preparation
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The following steps were performed under a UV-decontaminated Laminar Flow PCR workbench (STARLAB International GmbH, Hamburg, Germany), sterilized with DNA AWAY (Thermo Fisher Scientific, Waltham, MA, USA). Prior to library preparation, the amplified DNA was cleaned with DNA Clean & Concentrator—5 (Zymo Research, Irvine, CA, USA). DNA input for library preparation was normalized to 5.98 ng µL−1. Libraries were prepared using the NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina (New England Biolabs (NEB), Ipswich, MA, USA), following the <100 ng input protocol. Fragmentation was set to 14 min and 7 PCR cycles were used. NEBNext® Multiplex Oligos for Illumina® were used for barcoding. Library concentration and size was quantified with Qubit™ DNA HS assay (Life Technologies, Carlsbad, CA, USA) and a Bioanalyzer High Sensitivity DNA kit (Agilent, Santa Clara, CA, USA). The libraries were sequenced using an Illumina NextSeq 550 with the High Output Kit v2.5 300 Cycles (2 × 150 bp paired-end) (Illumina, San Diego, CA, USA).
5
Targeted Capture NGS for Inborn Errors of Metabolism
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Venous blood sample was used for DNA extraction using the QIAamp DNA Mini Kit (Qiagen, Germany). Targeted capture NGS was performed using Ion Ampliseq Inborn Errors of Metabolism Research Panel V2 (Life Technologies). The capture panel consisted of 9681 amplicons (1.05 Mbp) covering coding regions and flanking exon/intron boundaries of 594 genes associated with inborn errors of metabolism with an average gene coverage of 99.17%. Amplicon length and average insert size were 254 bp and 207 bp respectively. The library preparation for targeted capture was carried out according to the manufacturer’s protocol. Briefly, 100 ng of DNA quantified by Qubit DNA HS assay (Life Technologies) was used. The amplicons were then ligated to the barcode-labelled X adapter and the Ion Sphere Particle (ISP) compatible P adapter. The library was purified with AMPure XP Beads (Beckman Coulter, Pasadena, CA) and the final library concentration was measured using the Ion Library TaqMan® Quantitation Kit (Life Technologies). Following pooling equimolar libraries, automated template preparation and chip loading were carried out using Ion Chef System (Life Technologies). The Ion Proton Sequencing 200 kit (Life Technologies) was used for sequencing on a PI chip V2 following the manufacturer’s protocol.
6
ChIP-seq Protocol for ICP4 Protein
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Chromatin immunoprecipitation (ChIP) was performed as previously described [59 (link), 60 (link)] with the following modifications. Per sample, 40 μg of crosslinked DNA (as determined after reverse crosslinking, protease treatment, phenol extraction, and ethanol precipitation) was used as input. ICP4-3xflag crosslinked to chromatin was precipitated by incubation of sheared chromatin with anti-flag M1 agarose affinity gel (Sigma-Aldrich, catalog number A2220) for 3 h at 4 °C. Yields in the eluates were about 3 ng DNA for control samples (untransfected cells) and 10 ng for ICP4-ChIP samples, as determined by measurements with the Qubit DNA HS assay (Thermo Fisher, catalog number Q32851). For qPCR, 10 ng of DNA template for input and 50 pg for eluate samples was used, as described in the “RT-qPCR analysis” section above.
7
RNA Isolation and Sequencing
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Libraries were prepared using RNA of cells treated either with the vehicle or with Sfb. Total RNA was prepared as described above. Then, concentration and quality of the RNA were assessed with Qubit (Qubit™ DNA HS assay, Thermo Fisher Scientific, Waltham, MA, USA) and a 2100 Bioanalyzed Nano Chip (Agilent Technologies Genomics, Santa Clara, CA, USA), respectively. RNA Integrity Number (RIN) values were > 9 in all RNA samples. Polyadenylated RNA was isolated from the total RNA using NEBNext Oligo d(T)25 beads (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. Samples were normalized to an equivalent concentration of 67.3 ng/µL and prepared for RNA Ilumina Sequencing. For HepG2 cell lines, three biological replicates were obtained, while for SNU423 cells only two biological replicates were collected.
8
Whole Genome Sequencing of 339 Samples
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Total genomic DNA from the 339 samples was sequenced using the Illumina NextSeq 500 platform. Briefly, following total DNA quantification (Qubit DNA HS assay; Thermo Fisher Scientific), the sequencing libraries were generated using a Nextera XT library preparation kit (Illumina), according to the manufacturer’s instructions. The libraries were sequenced using NextSeq 500/550 mid output reagent cartridge v2 (300 cycles). Paired-end 150 bp reads were generated and their quality was assessed with FastQC [39 ]. Reads were trimmed with Trimmomatic version 0.36 and subsequently used to assemble genome scaffolds using SPAdes version 3.11.1 [40, 41 (link)].
9
Transcriptome Profiling of Chicken Neural Crest
10
Whole-Genome Sequencing of Beaver
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We performed whole-genome, short read sequencing at TCAG on the Illumina HiSeq X platform. Purified genomic DNA from fresh beaver blood leukocytes was quantified by Qubit DNA HS Assay (ThermoFisher). Genomic libraries were constructed using the Illumina Nano DNA Library Preparation Kit from DNA sheared using a Covaris LE220 Focused Ultrasonicator (Covaris, Woburn, MA) to an average length between 300 and 400 bases. We used 100 ng of sheared DNA for library preparation, following Illumina’s recommendation, and six amplification cycles. Libraries were quantified for sample loading using the KAPA Library Quantification Kit (Roche Diagnostics). Four lanes of DNA sequences (150 base paired-end reads of a 300–400 base insert) were produced, representing an estimated 160-fold coverage of the genome.
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