Fronds grown on inorganic nitrogen or on organic nitrogen medium were harvested and immediately frozen in liquid nitrogen. For each condition three biological replicates were analyzed. Total RNA was extracted by grinding 200 mg frozen tissue to a fine powder in liquid nitrogen with a mortar and pestle, followed by extraction with TRIzol Reagent (Invitrogen, MA, USA) and ethanol precipitation. Total RNA was resuspended and treated with RQ1 RNase-free DNase (Promega,WI, USA) on an RNA Clean & Concentrator-5 column (Zymo, CA, USA). The purity and concentration of RNA were determined by a NanoDrop Nd-1000 spectrophotometer (Thermo Scientific, MA, USA) and a Qubit fluorometer (Qiagen, MD, USA). Polyadenylated RNA was enriched from total RNA with the Dynabeads mRNA Purification Kit (Life Technologies, CA, USA). Indexed, directional RNA-seq libraries were prepared with ScriptSeq v2 reagents (Epicentre, WI, USA) from 40–50 ng of polyA-selected RNA according to the manufacturer’s instructions. Libraries were sequenced on an Illumina HiSeq 2000 instrument (Illumina, Inc., CA, USA) generating 101 bp paired-end reads. Reads from each sample were mapped against the 21,830 predicted transcripts of the Lemna gibba draft genome v0.5.1 [80 ] using Bowtie [81 (link)].
Qubit fluorometer
Manufactured by Qiagen
Sourced in United States
The Qubit fluorometer is a compact and sensitive instrument designed for accurate quantification of nucleic acids and proteins. It utilizes fluorescence-based detection to provide precise measurements of sample concentrations. The Qubit fluorometer is a compact and sensitive instrument designed for accurate quantification of nucleic acids and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Purelink rna mini column, by Thermo Fisher Scientific (2 mentions)
Trizol, by Zymo Research (2 mentions)
Tissuelyser, by Qiagen (2 mentions)
Novaseq 6000 system, by Illumina (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Hiseq 2000 instrument, by Illumina (1 mentions)
Dynabeads mrna purification kit, by Thermo Fisher Scientific (1 mentions)
Rna clean concentrator 5 column, by Zymo Research (1 mentions)
Rq1 rnase free dnase, by Promega (1 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (1 mentions)
Dnase and rnase free water, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna blood midi kit, by Qiagen (1 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions)
Rneasy plus kit, by Qiagen (1 mentions)
Allprep dna rna mini kit, by Qiagen (1 mentions)
Truseq stranded mrna kit, by Illumina (1 mentions)
Gentra blood kit, by Qiagen (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
12 protocols using qubit fluorometer
1
Transcriptome Analysis of Lemna gibba
2
Extraction and Purification of RNA from Mosquitoes and Nematodes
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Mosquito thoraces were combined with TRIzol (Zymo Research, Irvine, CA, USA) at a ratio of 1 mL TRIzol per 50–100 mg mosquito tissue while nematode samples were processed using a 3:1 volume ratio of TRIzol to sample. β-mercaptoethanol was added to a final concentration of 0.1%. The tissues were homogenized in a TissueLyser (Qiagen, Germantown, MD) at 50 Hz for 5 min. The homogenate was transferred to a new tube and centrifuged at 12,000 × g for 10 min at 4 °C. After incubating at room temperature for 5 min, 0.2 volumes of chloroform were added. The samples were shaken by hand for 15 s, incubated at room temperature for 3 min, then loaded into a pre-spun, phase lock gel heavy tube (5Prime, Gaithersburg, MD, USA) and centrifuged for 5 min at 12,000 × g at 4 °C. The upper phase was removed to a new tube and one volume of 100% ethanol was added prior to loading onto a PureLink RNA Mini column (Ambion, Austin, TX). The samples were then processed following manufacturer instructions, quantified using a Qubit fluorometer (Qiagen, Germantown, MD, USA) and/or a NanoDrop spectrometer (NanoDrop, Wilmington, DE, USA). The RNA was subsequently treated with the TURBO DNA-free kit (Ambion, ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.
3
Nucleic Acid Extraction and Quantification
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Bacterial genomic DNA (gDNA) and RNA was extracted using QIAamp One-For-All kit (QIAGEN) according to the manufacturer’s instructions and quantified using a Qubit™ fluorometer. Aliquots of nucleic acids were transported on ice to the clinical diagnostics laboratory for reverse-transcriptase-PCR (RT-PCR) analysis.
4
Quantitative analysis of ALLO 501A CART
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VCN analysis was performed and analyzed by Navigate Biosciences (Carlsbad, CA) utilizing a validated qPCR assay. Genomic DNA was extracted from patient peripheral blood and bone marrow aspirate samples collected in K2EDTA tubes utilizing the QIAamp Blood DNA Midi Kit (QIAGEN, Germantown, MD) and quantified using Qubit Fluorometer. Patient DNA input ranged from 20 to 200 ng per reaction and was analyzed with a validated primer probe set specific for the ALLO 501A CART drug product, 2X TaqManGene Expression Master Mix (Thermo Fisher Scientific, Waltham, MA) and molecular-grade water, ultrapure distilled water, and DNase and RNase-free water (Thermo Fisher Scientific). Analysis was conducted on the Applied Biosystems ViiA7 qPCR system in conjunction with the ViiA7 Applied Biosystems software version 1.2.4 or newer (Thermo Fisher Scientific).
5
Transcriptome Analysis by RNA-sequencing
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The transcriptomes were analysed with RNA-sequencing. Total RNA was extracted with the Rneasy Plus Kit (Qiagen, Valencia, CA) or the AllPrep DNA/RNA Mini Kit (Qiagen, Valencia, CA), and concentrations were measured by the Qubit fluorometer (RRID:SCR_018095). RNA integrity was confirmed with Agilent 2100 Bioanalyzer (RRID:SCR_018043). Sequencing libraries were prepared from 500 ng of total RNA with Illumina TruSeq stranded mRNA-kit. Sequencing was performed on the Illumina Novaseq 6000 system (RRID:SCR_016387) on two separate runs.
6
Genomic DNA Extraction and Sequencing
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The GENTRA Blood kit (Qiagen N.V.) was used for the isolation of genomic DNA from EDTA blood samples. The quantification and quality of the obtained DNA were assessed using the Qubit fluorometer (Qiagen N.V.). DNA was paired-end sequenced (read length of 150 base pair) as single-indexed genomic libraries using the Illumina Novaseq6000 (Illumina Inc., USA). Finally, raw reads were preprocessed by trimming the adapter sequences and removing the reads with 50% of low-quality nucleotides and fewer than 36 base pairs in length with fastp v0.23.1 [26 (link)].
7
RNA Extraction from Frozen Tissue
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Isolation was performed as previously described (Hocker et al. 2013 ) with modifications. Tissue samples were frozen in liquid nitrogen and stored at −80°C until use. A quantity of 5–20 mg of tissue was weighed and homogenized in 700 μL Qiazol (Qiagen, Germantown, MD) on ice using a Heidolph Silent Crusher (Schwabach, Germany). Nucleic acid extraction was performed using 140 μL of chloroform, the resulting aqueous layer was precipitated in 0.5 mL of isopropanol, and the resulting pellet was rinsed in ethanol, and resuspended in RNase‐free water. RNA quantification was performed using a Qubit fluorometer (Qiagen) and RNA quality was determined using a Fragment Analyzer (Advanced Analytical, Ames, IA). We set a RNA Quality Number (RQN) value cutoff of 5.7 for each sample.
8
Insect Gut Microbiome Metagenomics
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The surface of the insects was disinfected with 70% ethanol before dissecting the whole guts under a binocular microscope. Ten guts per termite species were pooled and placed in tubes containing RNA-later (Ambion, Grand Island, USA).
For DNA shotgun sequencing, total genomic DNA was isolated using the DNeasy Blood and Tissue kit (Qiagen, USA) following the manufacturer’s instructions. Crude DNA extracts were further purified according to the QIAmp DNA micro protocol for tissues (Qiagen, USA). The DNA concentration was determined with a Qubit fluorometer (Qiagen) and the size range assessed using a bioanalyzer and gel electrophoresis. The extracted DNA from each sample was diluted to a final concentration of 200 ng for the preparation of a metagenomic library at the QB3 Vincent J. Coates Genomics Sequencing Laboratory (Berkeley, USA). Briefly, DNA was sheared using Covaris and the library preparation was performed using the Kapa Biosystems Library Prep system on the IntegenX Apollo 324 robot. Each sample was sequenced using an Illumina platform (HiSeq 2500 Rapid Run) to generate 150 bp paired-ends reads.
For DNA shotgun sequencing, total genomic DNA was isolated using the DNeasy Blood and Tissue kit (Qiagen, USA) following the manufacturer’s instructions. Crude DNA extracts were further purified according to the QIAmp DNA micro protocol for tissues (Qiagen, USA). The DNA concentration was determined with a Qubit fluorometer (Qiagen) and the size range assessed using a bioanalyzer and gel electrophoresis. The extracted DNA from each sample was diluted to a final concentration of 200 ng for the preparation of a metagenomic library at the QB3 Vincent J. Coates Genomics Sequencing Laboratory (Berkeley, USA). Briefly, DNA was sheared using Covaris and the library preparation was performed using the Kapa Biosystems Library Prep system on the IntegenX Apollo 324 robot. Each sample was sequenced using an Illumina platform (HiSeq 2500 Rapid Run) to generate 150 bp paired-ends reads.
9
RNA Isolation from Cell Samples
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Cells collected for RNA isolation were resuspended with 1 mL of TRIzol reagent (Invitrogen, cat no: 15596018, ThermoFisher Scientific) and frozen until RNA extraction. The TRIzol-cell mixture was layered onto a Phasemaker Tube (Invitrogen, ThermoFisher Scientific, cat no: A33248) and incubated at room temperature for 3 min followed by addition of 200 µL chloroform (MP Biomedicals, ThermoFisher Scientific, catalog no: ICN19400280) and spun at 16,000g at 4 °C for 15 min to allow separation. The aqueous layer was recovered, 500 µL 70% ethanol was added, and then transferred a RNeasy Mini Kit (Qiagen, ThermoFisher Scientific, cat no: 74104) spin column and proper buffer washing steps were carried out following manufacturer recommendations including a 15 min on-column DNAse (Qiagen, ThermoFisher Scientific, cat no: 79254) treatment. RNA concentration was then quantified via Qubit Fluorometer using the RNA BR Assay Kit (Qiagen, ThermoFisher Scientific, cat no: Q10210) and quality was evaluated using an Agilent 2100 BioAnalyzer (Agilent, cat no: G2939BA).
10
Diverse Biological Sample Preparation
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The cell-free trout DNA (cfDNA)used for this experiment was extracted from frozen rainbow trout liver with chloroform phenol extraction [17 ] and then sonicated to create 400–600 bp fragments. The sonicated DNA was quantified by Qubit fluorometer (Qiagen, Manchester, UK) and stored at – 20 °C.
Mouse embryonic fibroblasts were removed from culture flasks by trypsinisation and washed in phosphate-buffered saline (PBS) pH 8.0 by centrifugation. The cells were then DAPI stained, and their nuclei were counted in a haemocytometer to determine their concentration. This was followed by cell resuspension at a concentration of 1 × 106/ml in PBS 20 % glycerol. The resuspended cells were stored at – 20 °C until required. Prior to use, the cells were centrifuged and re-suspended in Tris-buffered saline (TBS) to the required concentration [18 ].
Human saliva was collected from a volunteer and used fresh within 10 min of collection.
Bovine blood was sourced from a local abattoir (ABP Perth, Inveralmond Industrial Estate, Ruthvenfield Road, Perth UK) and treated with 12.5 % (v/v) of anticoagulant ACD immediately after sample collection. The blood was divided into aliquots which were frozen and stored at – 20 °C until required.
Mouse embryonic fibroblasts were removed from culture flasks by trypsinisation and washed in phosphate-buffered saline (PBS) pH 8.0 by centrifugation. The cells were then DAPI stained, and their nuclei were counted in a haemocytometer to determine their concentration. This was followed by cell resuspension at a concentration of 1 × 106/ml in PBS 20 % glycerol. The resuspended cells were stored at – 20 °C until required. Prior to use, the cells were centrifuged and re-suspended in Tris-buffered saline (TBS) to the required concentration [18 ].
Human saliva was collected from a volunteer and used fresh within 10 min of collection.
Bovine blood was sourced from a local abattoir (ABP Perth, Inveralmond Industrial Estate, Ruthvenfield Road, Perth UK) and treated with 12.5 % (v/v) of anticoagulant ACD immediately after sample collection. The blood was divided into aliquots which were frozen and stored at – 20 °C until required.
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