Splenic DC RNA quality was assessed via bioanalyzer (Agilent) and quantified via Qubit fluorometric quantification (Thermo Fisher). RNA-seq libraries were generated using 200ng total RNA as input for the TruSeq Stranded mRNA Library Prep kit (Illumina) according to the manufacturer’s protocol, and samples were indexed using TruSeq RNA Single Indexes (Illumina). Library preps were analyzed via bioanalyzer (Agilent) and quantified via Qubit fluorometric quantification (Thermo Fisher). Quantified libraries were normalized, pooled, and sequenced on a NextSeq 500 sequencer (Illumina) using the single-end 75 nucleotide setting. Raw sequencing reads were demultiplexed, and FASTQ files were aligned to the mouse genome (mm10) via Tophat v2.1.1 and Bowtie v2.3.2. BAM files were indexed with Samtools v1.6 and annotated using Partek software v7.17.0918 to generate RPKM values for each gene.
Qubit fluorometric quantification
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The Qubit Fluorometric Quantification is a lab equipment product that provides accurate and sensitive measurement of DNA, RNA, and protein concentrations. It utilizes fluorescence-based detection technology to quantify the target biomolecules.
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Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (5 mentions)
Nextseq 500 sequencer, by Illumina (4 mentions)
Bioanalyzer, by Agilent Technologies (4 mentions)
Dneasy blood and tissue kit, by Qiagen (4 mentions)
Truseq rna single indexes, by Illumina (3 mentions)
Truseq stranded mrna library prep kit, by Illumina (3 mentions)
Novaseq, by Illumina (3 mentions)
Tapestation, by Agilent Technologies (3 mentions)
Tapestation system, by Agilent Technologies (3 mentions)
Ampure xp bead, by Beckman Coulter (3 mentions)
Nucleospin tissue kit, by Macherey-Nagel (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Fragment analyzer, by Agilent Technologies (2 mentions)
Genejet pcr purification kit, by Thermo Fisher Scientific (2 mentions)
Nebnext ultra 2 dna library prep kit, by Illumina (2 mentions)
Agilent 2200 tapestation d100 high sensitivity kit, by Agilent Technologies (2 mentions)
Phix174 control dna, by Illumina (2 mentions)
Iscan system, by Illumina (2 mentions)
Infinium methylationepic beadchip, by Illumina (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
78 protocols using qubit fluorometric quantification
1
Splenic DC Transcriptome Profiling
2
Splenic DC Transcriptome Profiling
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Splenic DC RNA quality was assessed via bioanalyzer (Agilent) and quantified via Qubit fluorometric quantification (Thermo Fisher). RNA-seq libraries were generated using 200ng total RNA as input for the TruSeq Stranded mRNA Library Prep kit (Illumina) according to the manufacturer’s protocol, and samples were indexed using TruSeq RNA Single Indexes (Illumina). Library preps were analyzed via bioanalyzer (Agilent) and quantified via Qubit fluorometric quantification (Thermo Fisher). Quantified libraries were normalized, pooled, and sequenced on a NextSeq 500 sequencer (Illumina) using the single-end 75 nucleotide setting. Raw sequencing reads were demultiplexed, and FASTQ files were aligned to the mouse genome (mm10) via Tophat v2.1.1 and Bowtie v2.3.2. BAM files were indexed with Samtools v1.6 and annotated using Partek software v7.17.0918 to generate RPKM values for each gene.
3
DNA Extraction and Evaluation Protocol
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MicroLYSIS®-PLUS (Microzone Ltd., Haywards Heath, West Sussex, United Kingdom) was used to release DNA from single colonies of each of the ACC positive and 18 ACC negative isolates following the manufacturer’s protocol and utilized as the template DNA in polymerase chain reaction (PCR) reactions for amplifying the and acdS gene fragments. These pseudomonad isolates were selected for full genome sequencing, in which high quality genomic DNA was extracted from 10 mL LB cultures of each isolate using the GenElute genomic DNA extraction kit (Sigma-Aldrich), as per the manufacturer’s protocol. DNA quantity was determined using the Qubit Fluorometric Quantification (Life Technologies) using the manufacturer’s instructions. DNA quality was determined using the NanoDrop Microvolume UV spectrophotometer (Thermo Fisher Scientific Inc.) by OD at 260 nm, the 260/280 ratio was used to determine DNA quality in addition to running the samples on a 1.5% (w/v) agarose gel in 1 × Tris-borate-EDTA (TBE) stained with EtBr (0.2 μg mL–1) with TBE as the running buffer. Bands of DNA were viewed under UV light to visually detect smears arising from any degraded DNA.
4
Fungal ITS2 Amplicon Sequencing
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DNA extracted from swabs was used to amplify the ITS2 region of the fungal internal transcribed spacer (ITS) using custom barcoded primers (Kozich et al., 2013 (link)) and the following PCR conditions: denaturing step of 95°C for 2 min, followed by 35 cycles of 95°C for 20 s, 50°C for 20 s, 72°C for 5 min and a final extension step of 72°C for 5 min. Each PCR plate included a negative control and was performed in duplicate. Amplicons were visualized on a 1.5% agarose gel and pooled yielding a final per sample volume of 50 μL. Pooled amplicon DNA was purified using AMPure XP bead clean-up (Beckman Coulter, California, USA). Qubit fluorometric quantification (Life Technologies, California, USA) was used to determine the concentration of each purified sample, which were equimolar pooled to create the final library sample. This pooled sample was run on an Agilent 2200 TapeStation system (Agilent Technologies, California, USA) to assess amplicon distribution and presence of primer dimer. The sample underwent 300 bp paired-end sequencing using V3 chemistry on an Illumina MiSeq platform.
5
Isolation and Quantification of Primary Leaf RNA
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The frozen primary leaves were homogenized with a tube pestle (Biozym) in liquid nitrogen. Total RNA from the primary leaves was isolated with the InviTrap Spin Plant RNA Mini Kit (STRATEC Molecular), using lysis solution RP and following the manufacturer’s instructions. After incubation for 15 min at room temperature, an additional incubation for 3 min at 55 °C was conducted to get a higher RNA yield. Total RNA yield was measured by Qubit fluorometric quantification (Life technologies) and concentration was adjusted to 50 ng. RNA was used for cDNA synthesis with the QuantiTect Reverse Transcription Kit (Qiagen) following the manufacturer’s instructions. cDNA was stored at −20 °C.
6
Protein Extraction and Digestion Protocol
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At the end of the exposure period, we discarded the culture medium and carefully washed the cells 3 × with ice-cold PBS. Next, 1 mL of ice-cold PBS plus protease inhibitor (ProtoSTOP, Roche) was added to the plates and the cells were harvested with the aid of a cell scraper. We centrifuged the cell suspensions for 5 min at 600 g and discarded the supernatant. Cell pellets were stored at −80 °C until further analysis.
We resuspended cell pellets in lysis buffer (6 M urea, 2 M thiourea, protease inhibitors, 20 mM triethylammonium bicarbonate, and 10 mM 1,4-dithiothreitol reducing agent) at room temperature for 2 h. Then, the urea concentration was diluted 10 × and the cell lysis was enhanced by tip sonication on ice. We quantified proteins using Qubit fluorometric quantification (LifeTechnologies) and alkylated 50 µg of proteins in 20 mM iodoacetamide for 30 min in the dark. Following incubation, proteins were digested with trypsin (50:1 w/w protein:trypsin) overnight at room temperature. We acidified the peptide solution with 1% v/v formic acid to stop trypsin digestion and dried the peptides prior to desalting.
We resuspended cell pellets in lysis buffer (6 M urea, 2 M thiourea, protease inhibitors, 20 mM triethylammonium bicarbonate, and 10 mM 1,4-dithiothreitol reducing agent) at room temperature for 2 h. Then, the urea concentration was diluted 10 × and the cell lysis was enhanced by tip sonication on ice. We quantified proteins using Qubit fluorometric quantification (LifeTechnologies) and alkylated 50 µg of proteins in 20 mM iodoacetamide for 30 min in the dark. Following incubation, proteins were digested with trypsin (50:1 w/w protein:trypsin) overnight at room temperature. We acidified the peptide solution with 1% v/v formic acid to stop trypsin digestion and dried the peptides prior to desalting.
7
Amplification and Sequencing of Ancora sagittata Genome
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The DNA sample extracted from the infected cells of Ancora sagittata WSBS2006 was amplified with the REPLI-g Midi kit (QIAGEN) following the manufacturers protocol. Two amplification reactions were performed with an estimated 1 ng of starting material, which yielded 10 and 23 μg of DNA. DNA quantity assessments were performed with Qubit fluorometric quantification (Life Technologies). The amplified DNA samples were used to prepare two sequencing libraries for the Illumina platform. Paired-end libraries with estimated insert lengths of 333 bp (41 bp SD) and 424 bp (63 bp SD) were constructed using TruSeq library preparation protocol (Illumina) and sequenced on a HiSeq 2000 instrument. A total of 38M and 44M 100-bp paired end reads were obtained for the two libraries, respectively. The reads were adapter trimmed with Trimmomatic v0.30 in Paired End mode (Bolger et al. 2014 (link)), requiring a minimal length of 55 bp for each read to keep the pair.
8
Western Blot Analysis of Prestin Protein
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Western blot analyses were performed according to Yu et al. (2011 (link)) with slight modifications. The cochleae were lysed in a lysis buffer (5 mM Tris pH 6.8, 2% SDS, 2 mM EDTA, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/mL aprotinin, 2 µg/mL leueptine, 1 mM vanadate, 10 mM sodium fluoride and 20 mM β-glycerophosphate) for 1 h at 4°C with gentle shaking and then centrifuged at 13000 × g for 15 min to eliminate debris. Protein concentration was quantified using Qubit Fluorometric quantification (Life Technologies). Lysates were added 5x SDS buffer and boiled for 6 min to denature the proteins. Proteins (20 µg/sample) were resolved by 7.5% SDS–PAGE and Western blots were performed with antibodies against the different proteins. Blots were developed in WesternBrigth ECL detection kit (Advansta). Films were digitized using an Epson V750 scanner.
Antibodies used were anti-Prestin (1:500) made in rabbit, a kind gift from Dr. Bechara Kachar (Laboratory of Cell Structure and Dynamics, National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, Maryland), anti-beta tubulin (1:4000) and anti-actin (1:4000) from Sigma.
Antibodies used were anti-Prestin (1:500) made in rabbit, a kind gift from Dr. Bechara Kachar (Laboratory of Cell Structure and Dynamics, National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, Maryland), anti-beta tubulin (1:4000) and anti-actin (1:4000) from Sigma.
9
Comparative Genomics of Bacterial Isolates
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DNA was extracted from isolates grown in MAG broth incubated at 28 °C and shaken at 100 rpm using the GenElute Bacterial Genomic DNA kit (Sigma Aldrich, USA). Extracted DNA was quantified using Qubit Fluorometric Quantification (Life Technologies, USA). Two sequencing platforms were used; Ion Torrent PGM™ (Life Technologies, USA) (located at Rothamsted Research, UK) and Illumina HiSeq 2000 (Illumina Inc, USA) (located at BGI, the Beijing Genomics Institute, China). For Ion Torrent sequencing, two barcoded, unamplified sequencing libraries were constructed using an Ion Plus Fragment Library Kit (Life Technologies, USA) using 1 μg template DNA. Libraries were pooled and sequence template generated using an Ion PGM™ Template OT2 400 Kit. Sequencing was performed using the Ion PGM™ Sequencing 400 Kit and an Ion 318™ Chip. The G22 sample generated 3,652,179 reads and the BF49 sample generated 3,129,252 reads. Illumina sequencing was performed by BGI and 40 ug of genomic DNA was used. A 6 kb mate library with 90 bp read length and 50X coverage was created using the Illumina HiSeq 2000 sequencing platform resulting in 2,997,699 paired reads for G22 and 3,026,005 paired reads for BF49.
10
Genomic DNA Extraction and Sequencing
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Total genomic DNA (gDNA) was extracted from the tissue samples using a NucleoSpin Tissue kit (Macherey‐Nagel) according to the manufacturer's protocol. The quality of the total gDNA was assessed using a Fragment Analyzer (Advanced Analytical), and the concentration was measured using Qubit fluorometric quantification (Life Technologies). Sequencing libraries (average insert size 350 bp) were constructed from 1 µg of gDNA for each individual according to the Illumina TruSeq DNA PCR‐Free Library Preparation Guide (part #15036187). A unique Illumina TruSeq indexing adapter was ligated to each gDNA sample. The samples were normalized and pooled for automated cluster preparation using an Illumina cBot station. Sixteen libraries (each corresponding to one perch sample from Estonia) were pooled and sequenced in eight lanes on an Illumina HiSeq 3000 using paired‐end sequencing (2 × 150‐bp read length with 8‐bp index), and another 16 libraries (each corresponding to one perch sample from Finland, Lithuania and Sweden) were pooled and sequenced in four lanes on an Illumina NovaSeq 6000 using paired‐end sequencing (2 × 150‐bp read length with 8‐bp index).
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