Qubit fluorometry

Manufactured by Thermo Fisher Scientific

Sourced in United States, Canada, Germany

The Qubit fluorometer is a sensitive and precise instrument used for quantifying nucleic acids (DNA, RNA) and proteins in sample solutions. It utilizes fluorescent dyes that bind specifically to the target molecules, allowing for accurate and reproducible concentration measurements.

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Lab products found in correlation

Bioanalyzer dna 1000 chip, by Thermo Fisher Scientific (11 mentions) Hiseq 4000, by Illumina (10 mentions) Truseq rna sample prep kit v2, by Illumina (8 mentions) Hiseq 2000, by Illumina (7 mentions) Ampure xp bead, by Beckman Coulter (6 mentions) Bullet blender, by Next Advance (6 mentions) Nanodrop, by Thermo Fisher Scientific (6 mentions) Hiseq 2500 system, by Illumina (5 mentions) Ribo zero rrna removal kit human mouse rat, by Illumina (5 mentions) Hiseq 2000 sequencer, by Illumina (5 mentions) Bioanalyzer, by Thermo Fisher Scientific (4 mentions) Trypsin, by Promega (4 mentions) Nanodrop spectrophotometry, by Thermo Fisher Scientific (4 mentions) Cbot and hiseq 3000 4000 pe cluster kit, by Illumina (4 mentions) Hiseq 3000 4000 pe cluster kit, by Illumina (4 mentions) Trizol reagent, by Thermo Fisher Scientific (4 mentions) Gentra puregene blood and tissue extraction kit, by Qiagen (4 mentions) Truseq rna sample prep kit v2 protocol, by Illumina (3 mentions) Dneasy blood and tissue kit, by Qiagen (3 mentions) Hiseq 2500, by Illumina (3 mentions)

Close spelling variants of this product

Qubit 2.0 fluorometry (8 citations) Qubit fluorometry assay (4 citations) Qubit 3.0 fluorometry (5 citations)

94 protocols using qubit fluorometry

RNA-seq Library Preparation and Sequencing for Plasma Cells

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RNA was extracted from CD138+ plasma cells using Qiagen’s RNeasy mini kit. Total RNA concentration and quality were determined using Qubit fluorometry (Invitrogen) and the Agilent Fragment Analyzer. Using the Illumina TruSeq® RNA Exome Library Prep kiT, libraries were prepared according to the manufacturer’s instructions. The concentration and purity of cDNA libraries were checked using the Agilent TapeStation D1000. Coding regions of the transcriptome were captured by pooling four of the cDNA libraries at 200 ng each. The concentration and size distribution of the completed libraries were determined using an Agilent BioAnalyzer DNA 1000 chip and Qubit fluorometry (Invitrogen). Libraries were sequenced at up to eight samples per lane following Illumina’s standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 100 × 2 paired-end reads on an Illumina HiSeq 4000 using the HiSeq Control Software HD 3.4.0.38 collection software. Base-calling was performed using Illumina’s RTA version 2.7.7.

Visram A., Dasari S., Anderson E., Kumar S, & Kourelis T.V. (2021). Relapsed multiple myeloma demonstrates distinct patterns of immune microenvironment and malignant cell-mediated immunosuppression. Blood Cancer Journal, 11(3), 45.

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Amplicon Sequencing Library Preparation

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Dfr1 amplicons prepared by PCR were first purified using the Thermo Fisher Scientific PCR purification kit, according to the manufacturer’s instructions, quantified using Qubit fluorometry (Life Technologies) and diluted for sequencing library preparation. Libraries were constructed using plexWell library kit technology (seqWell, Beverly MA). In this approach, each 1+ kb pool of diverse amplicons is tagged with a pool-specific barcode via a transposase-mediated adapter addition at random locations. After this tagging, the pools of amplicons are then pooled into a single meta-pool, and subjected to a second transposase-mediated adapter addition. Fragments of this pool containing sequence from each of the two iterative adapter additions are then amplified to yield a final sequence library representing identifiable fragments from each original amplicon pool. Sequencing data is available upon request. We have deposited the raw fastq files at the NCBI SRA under the accession number SRP072709.

Wong L.H., Sinha S., Bergeron J.R., Mellor J.C., Giaever G., Flaherty P, & Nislow C. (2016). Reverse Chemical Genetics: Comprehensive Fitness Profiling Reveals the Spectrum of Drug Target Interactions. PLoS Genetics, 12(9), e1006275.

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RNA-seq analysis of miR-155 knockout and Alzheimer's mouse models

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Total RNAs from PFC of 4- and 8-month-old male WT (n=4), miR155^+/− (n=4), miR155^−/−(n=4), APP/PSEN1 (n=4), APP/PSEN1;miR155^+/− (n=4) or APP/PSEN1;miR155^−/− (n=4) mice were subjected to RNA sequencing. The sequencing library was prepared with the TruSeq RNA Sample Prep Kit v2 protocol (Illumina, CA, USA). Ribosomal RNAs were removed using the Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat) (Illumina, CA, USA). Remaining RNAs were fragmented, and the cDNAs synthesized using random hexamers, end-repaired, and ligated with appropriate adaptors for sequencing. The library was processed for size selection and purification using AMPure XP beads (Beckman Coulter, CA, USA). The appropriate Illumina-recommended 6bp barcode bases were introduced at one end of the adaptors during the PCR amplification step. The size and concentration of the RNA-seq libraries were measured by Bioanalyzer and Qubit fluorometry (Life Technologies, NY, USA) before loading onto the sequencer. The rRNA-depleted libraries were sequenced on the Illumina HiSeq 2500 System with 100 nucleotide single-end reads (Illumina, CA, USA).

Readhead B., Haure-Mirande J.V., Mastroeni D., Audrain M., Fanutza T., Kim S.H., Blitzer R.D., Gandy S., Dudley J.T, & Ehrlich M.E. (2020). miR155 regulation of behavior, neuropathology, and cortical transcriptomics in Alzheimer’s disease. Acta neuropathologica, 140(3), 295-315.

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RNA-seq of Tyrobp-deficient APP/PSEN1 mouse brains

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Total RNAs from PFC of 8-month-old male WT (n=4), Tyrobp^+/− (n=3), Tyrobp^−/−(n=4), APP/PSEN1 (n=4), APP/PSEN1;Tyrobp^+/− (n=4) or APP/PSEN1;Tyrobp^−/− (n=5) mice were subjected to RNA sequencing. The sequencing library was prepared with the TruSeq RNA Sample Prep Kit v2 protocol (Illumina, CA, USA). Ribosomal RNAs were removed using the Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat) (Illumina, CA, USA). Remaining RNAs were fragmented, and the cDNAs synthesized using random hexamers, end-repaired, and ligated with appropriate adaptors for sequencing. The library was processed for size selection and purification using AMPure XP beads (Beckman Coulter, CA, USA). The appropriate Illumina-recommended 6bp barcode bases were introduced at one end of the adaptors during the PCR amplification step. The size and concentration of the RNA-seq libraries were measured by Bioanalyzer and Qubit fluorometry (Life Technologies, NY, USA) before loading onto the sequencer. The rRNA-depleted libraries were sequenced on the Illumina HiSeq 2500 System with 100 nucleotide single-end reads (Illumina, CA, USA).

Haure-Mirande J.V., Wang M., Audrain M., Fanutza T., Kim S.H., Heja S., Readhead B., Dudley J.T., Blitzer R.D., Schadt E.E., Zhang B., Gandy S, & Ehrlich M.E. (2018). Integrative approach to sporadic Alzheimer’s disease: deficiency of TYROBP in cerebral Aβ amyloidosis mouse normalizes clinical phenotype and complement subnetwork molecular pathology without reducing Aβ burden. Molecular Psychiatry, 24(3), 431-446.

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DNA Extraction of Rhizobium leguminosarum

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Rlv UPM791 is a spontaneous streptomycin-resistant derivative [17 (link)] of strain 128C53, a hydrogenase-positive [18 (link)] isolate from pea nodules received from J. Burton (Nitragin Co., Milwaukee, WI, USA). It was grown in tryptone yeast (TY) broth [19 (link)] at 28 °C during two days to carry out genomic DNA extraction. For 454 (Roche GS FLX, Roche, Basel, Switzerland) and Illumina (San Diego, CA, USA) platform sequencing, a Cetyl Trimethyl Ammonium Bromide (CTAB)-based protocol [20 (link)] was used, whereas for PacBio platform sequencing, DNA was purified with a DNA extraction blood tissue kit (Qiagen Hilden, Germany). DNA samples were quantified by Qubit fluorometry (Life Technologies, Carlsbad, CA, USA), and by Nanodrop spectrophotometry (Thermo Scientific, Waltham, MA, USA).

Sánchez-Cañizares C., Jorrín B., Durán D., Nadendla S., Albareda M., Rubio-Sanz L., Lanza M., González-Guerrero M., Prieto R.I., Brito B., Giglio M.G., Rey L., Ruiz-Argüeso T., Palacios J.M, & Imperial J. (2018). Genomic Diversity in the Endosymbiotic Bacterium Rhizobium leguminosarum. Genes, 9(2), 60.

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Sugar Beet Germplasm Genotyping Protocol

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Seeds from a total of 606 Beta accessions were obtained from the public germplasm repositories of the USDA (https://www.ars-grin.gov/) and the IPK (https://www.ipk-gatersleben.de/), from three sugar beet breeding companies (KWS SAAT SE, Syngenta, Strube Research GmbH), and from the USDA sugar beet breeding program at Michigan State University, East Lansing, MI, USA. All plants were grown in the greenhouse at KWS, and leaf material was harvested from one single plant per accession approximately six weeks after sowing. Plant genomic DNA for genotyping and sequencing was extracted by a silica-membrane method using the NucleoSpin 96 Plant II kit (Macherey-Nagel, Düren, Germany). Genotyping data were produced with the Illumina Infinium HD-chip workflow according to the manufacturer´s instructions (Illumina, San Diego, CA, USA). Following quantification of genomic DNA by Qubit fluorometry (Life Technologies, Foster City, CA, USA), 200 ng of each DNA sample was sheared to a peak size of 580 bp with a Covaris M220 instrument (Covaris, Woburn, MA, USA).

Wascher F.L., Stralis-Pavese N., McGrath J.M., Schulz B., Himmelbauer H, & Dohm J.C. (2022). Genomic distances reveal relationships of wild and cultivated beets. Nature Communications, 13, 2021.

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Genomic DNA Extraction and Sequencing of Pasteurella multocida

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Genomic DNA was purified from each of the twelve P. multocida strains using the Qiagen DNeasy blood and tissue kit (Qiagen Cat# 69504) using 5 mL of overnight cultures grown at 37°C in brain heart infusion (BHI) broth (Oxoid, UK) and following the Gram-negative bacterial protocol outlined in the manufacturer’s instructions. DNA quantification and purity analysis was performed by agarose gel electrophoresis and Qubit Fluorometry (Life Technologies, USA). The purified genomic DNA was sequenced using the paired-end 90-bp sequencing protocol on an Illumina HiSeq 2000 (Illumina Inc., San Diego, USA) at the Beijing Genomics Institute (BGI), China. The raw read sequences were filtered to eliminate low quality reads using the following criteria; all reads with > 40% low quality (Q20) bases (parameter setting at 36 bp), > 10% Ns (parameter setting at 9 bp) or > 15 bp overlap with Illumina TruSeq adapter sequences (parameter setting at 15 bp) were removed.

Moustafa A.M., Seemann T., Gladman S., Adler B., Harper M., Boyce J.D, & Bennett M.D. (2015). Comparative Genomic Analysis of Asian Haemorrhagic Septicaemia-Associated Strains of Pasteurella multocida Identifies More than 90 Haemorrhagic Septicaemia-Specific Genes. PLoS ONE, 10(7), e0130296.

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Hippocampal Cell Culture Proteomics

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Hippocampal cultures, prepared as above, were treated with either 1 pM or 30 nM NYX‐2925 for 30 min, also as described above (n = 4 independent cell culture preparations). Treated cultures were washed in cold PBS and frozen at −80°C until analysis. Subsequent sample handling, mass spectrometry, and data analysis were performed by MSBioworks LLC (Ann Arbor, MI, USA). Cells were lysed in 500 μL urea buffer (8 μM urea, 50 mM Tris HCl, 150 mM NaCl, 1X Roche (Indianapolis, IN, USA) Complete Proteinase Inhibitor (Sigma‐Aldrich, Cat #: 11836145001, pH 8.0). Samples were incubated at 20℃ for 1 h, clarified by centrifugation, and quantified by Qubit fluorometry (Life Technologies, Rockville, MD, USA). A 50 μg aliquot of each sample was digested in solution using the following protocol: sample was diluted in 25 mM ammonium bicarbonate (Cat #: A643), reduced with 10 mM dithiothreitol (Cat #: AAJ1539706) at 60°C, alkylated using 50 mM iodoacetamide (Cat #: AC122270050) at 20℃, digested with 2.5 μg trypsin (Promega, Madison, WI, USA, Cat #: V5280) at 37°C for 18 h followed by quenching with formic acid. Peptides were cleaned using solid‐phase extraction using the Empore C18 plate (3M, St. Paul, MN, USA, Cat #: 14‐378‐57).

Bowers M.S., Cacheaux L.P., Sahu S.U., Schmidt M.E., Sennello J.A., Leaderbrand K., Khan M.A., Kroes R.A, & Moskal J.R. (2019). NYX‐2925 induces metabotropic N‐methyl‐d‐aspartate receptor (NMDAR) signaling that enhances synaptic NMDAR and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor. Journal of Neurochemistry, 152(5), 523-541.

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Genomic DNA Extraction and Sequencing for BHBM Resistance

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Genomic DNA was extracted from RYO0622 and BHBM-resistant cells using a standard yeast DNA extraction protocol (60 (link)). Genomic DNA samples were quantified using Qubit fluorometry (Life Technologies) and diluted for sequencing library preparation using a Nextera XT library preparation kit according to the manufacturer’s instructions (Illumina, San Diego, CA). For the initial round of sequencing, individual sequencing libraries were prepared for the parent and a single BHBM-resistant clone. These libraries were pooled and sequenced on a single MiSeq lane (Illumina), generating paired-end 150-bp reads. Further BHBM-resistant colonies were obtained in a second screen, and their DNAs were pooled at equal concentrations before preparation of a single sequencing library for the pool. This pool was sequenced alongside a new library for the parent strain on a single HiSeq 2500 lane (Illumina), generating paired-end 100-bp reads.

Mor V., Rella A., Farnoud A.M., Singh A., Munshi M., Bryan A., Naseem S., Konopka J.B., Ojima I., Bullesbach E., Ashbaugh A., Linke M.J., Cushion M., Collins M., Ananthula H.K., Sallans L., Desai P.B., Wiederhold N.P., Fothergill A.W., Kirkpatrick W.R., Patterson T., Wong L.H., Sinha S., Giaever G., Nislow C., Flaherty P., Pan X., Cesar G.V., de Melo Tavares P., Frases S., Miranda K., Rodrigues M.L., Luberto C., Nimrichter L, & Del Poeta M. (2015). Identification of a New Class of Antifungals Targeting the Synthesis of Fungal Sphingolipids. mBio, 6(3), e00647-15.

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SILAC-Based Nuclear Proteome Analysis

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All SILAC sample preparation, mass spectrometry (MS), and MS data processing were performed by MS Bioworks (http://www.msbioworks.com/). To determine label incorporation, the concentrations of proteins that were labeled with heavy labeling media were determined using Qubit fluorometry (Life Technologies). 20 μg of sample was processed by SDS-PAGE on a 4-12% Bis-Tris Novex mini-gel (Life Technologies) using the MOPS buffer system. The gel was stained with InstantBlue (Expedeon) and bands were excised at 50 and 100 kDa. Gel bands were processed using a robot (ProGest, DigiLab) as follows: the bands were washed with 25 mM ammonium bicarbonate followed by acetonitrile, reduced with 10 mM dithiothreitol at 60°C followed by alkylation with 50 mM iodoacetamide at room temperature, digested with trypsin (Promega) at 37°C for 4 h, quenched with formic acid, and the supernatant was analyzed directly without further processing. For the nuclear proteome SILAC analysis, 10 μg each of heavy and light media-labeled nuclear fractions were combined and processed by SDS-PAGE using a 4-12% Bis Tris NuPage mini-gel (Life Technologies). Calibration was performed with Thermo PageRuler broad range markers. The mobility region was excised into forty equal sized segments using a grid. Each gel segment was processed as described above.

Justice J.L., Verhalen B., Kumar R., Lefkowitz E.J., Imperiale M.J, & Jiang M. (2015). Quantitative Proteomic Analysis of Enriched Nuclear Fractions from BK Polyomavirus-infected Primary Renal Proximal Tubule Epithelial Cells. Journal of proteome research, 14(10), 4413-4424.

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