For the variety “Fielder” and the progenies of gene editing, total DNA was extracted using a DNA extraction kit (Tiangen, Beijing, China). The quality and concentration of the total DNA were determined using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, United States). Total RNA was extracted using an AG21019 RNA extraction kit (Accurate, Changsha, China), digested with DNase to remove residual DNA, and reverse transcribed into cDNA using a Prime Script TMRT-PCR kit AG11711 (Accurate, Changsha, China). The quality and concentration of the total RNA were determined using 1% agarose gel electrophoresis and a NanoDrop 2000c spectrophotometer (Thermo, Wilmington, DE, United States). Genomic DNA was extracted using the Tiangen DNA quick Plant System (Tiangen, Beijing, China) for deep sequencing.
Nanodrop 2000c spectrophotometer
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany, United Kingdom, Spain, Canada, Italy, Switzerland, Japan, France, Australia, Lithuania, Denmark
The NanoDrop 2000c spectrophotometer is a compact and versatile instrument designed for the quantification and analysis of nucleic acids and proteins. It utilizes a patented sample retention system that allows for accurate measurements of small sample volumes, typically ranging from 0.5 to 2 microliters. The NanoDrop 2000c provides precise absorbance measurements across a wide wavelength range, enabling users to determine the concentration and purity of various biomolecules.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (198 mentions)
Rneasy mini kit, by Qiagen (87 mentions)
Trizol, by Thermo Fisher Scientific (70 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (54 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (46 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (44 mentions)
Primescript rt reagent kit, by Takara Bio (44 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (38 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (35 mentions)
2100 bioanalyzer, by Agilent Technologies (32 mentions)
Rneasy plus mini kit, by Qiagen (29 mentions)
Iscript cdna synthesis kit, by Bio-Rad (28 mentions)
Dneasy blood and tissue kit, by Qiagen (27 mentions)
Rneasy plant mini kit, by Qiagen (24 mentions)
Qiaamp dna mini kit, by Qiagen (23 mentions)
Mirneasy mini kit, by Qiagen (23 mentions)
Dneasy blood tissue kit, by Qiagen (20 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (19 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (18 mentions)
Rnaiso plus, by Takara Bio (18 mentions)
1 680 protocols using nanodrop 2000c spectrophotometer
1
Genetic Analysis for Gene-Edited Fielder
2
Plasmid Preparation and cRNA Synthesis for Aquaporin Expression
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Table 2 contains a list of the expression vectors, restriction enzymes and promoters used in this study. The plasmids encoding for hAQP1, hAQP1FLAG, hAQP1C189S mutant, rAQP3, hAQP7, hAQP8 or rAQP9 were transformed into TOP10 competent cells. All of the plasmids were sequenced using the BigDye kit and the ABI Prism 3130XL Genetic Analyzer (Hitachi, Tokyo, Japan). Plasmid DNA was purified using either miniprep or midiprep kits (Qiagen). DNA concentration and purity were determined spectrophotometrically on a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific).
Plasmids were linearized using the respective restriction enzyme (Table 2 ) and restriction-digested products were purified using the QIAquick PCR purification kit (Qiagen). The capped RNAs (cRNAs) were transcribed using either a T3 or T7 mMessage mMachine kit (Ambion, Austin, USA) depending on the promoter present on the linearized DNA (Table 2 ). The cRNA was then purified and concentrated using the RNeasy MinElute RNA Cleanup kit (Qiagen) and quantified by measuring the absorbance at 260 nm using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific).
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Plasmids were linearized using the respective restriction enzyme (
3
Comprehensive DNA and RNA Extraction
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Total DNA was extracted from primary fibroblast cells and mouse tissues using a DNeasy plus mini kit and DNeasy blood & tissue kit (Qiagen, Valencia, CA, United States) following the manufacturer’s instruction (Jasoliya et al., 2019 (link)). DNA was quantified by a NanoDrop 2000c Spectrophotometer (Thermo Scientific, Waltham, MA, United States). Total RNA was extracted from human fibroblast cells using an RNeasy plus mini kit (Qiagen, Valencia, CA, United States), following the manufacturer’s instructions. RNA quantity and quality were measured by using a NanoDrop 2000c Spectrophotometer (Thermo Scientific, Waltham, MA, United States). For mouse tissues, total RNA was isolated by homogenization with TRIzol (Invitrogen, United Kingdom).
4
Quantification of TRAP Proteins
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All dTRAP proteins shared the same predicted extinction coefficient at 280 nm (ε = 4470 M−1cm−1; ProtParam). Protein concentrations were measured using a Nanodrop 2000c spectrophotometer (Thermo Scientific) in droplet mode. Unless otherwise specified, TRAP concentrations refer to the concentration of the TRAP 12-mer ring, not the protomer concentration. Ring concentration is 1/12th the promoter concentration for WT TRAP; it’s 1/6th the protomer concentration for dTRAP and its variants. The binding site concentration equals the promoter concentration for WT TRAP, it’s twice the concentration for WT-WT dTRAP, and it equals the promoter concentration for Mut-WT and WT-Mut dTRAP. Trp concentration was determined by UV absorbance at 280 nm with the extinction coefficient ε =5540 M−1 cm−1 using a Nanodrop 2000c spectrophotometer (Thermo Scientific) in droplet mode.
5
Rifampin-Loaded HyStem Hydrogel for MRSA
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Rifampin (600 μg) was mixed with three different percentages of HyStem Hydrogel (0.5, 1, and 2%; ESI-BIO, Alameda, CA, USA) and then applied to polyester (PET) track-etched membrane inserts (3.0-μm pore size; Corning Incorporated Life Science, NY, USA) for 30 min in a biosafety cabinet. Afterward, inserts were transferred into 24-well plates with DMEM containing 10% FBS and 1% of a penicillin/streptomycin solution (Gibco) and then incubated in a humidified atmosphere containing 5% CO2 at 37°C (Thermo Fisher Scientific Inc.). Kinetic cumulative release of rifampin (absorbance = 336 nm) was measured by the NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific Inc.). Media containing rifampin (600 μg) was used as a positive control (100%). On days 1, 4, and 7, we washed the inserts with PBS solution at least three times and transferred them into LB media containing oxacillin (6 μg/ml) and MRSA (4 × 106 CFU). The inserts were then incubated for 24 hours. MRSA proliferation was measured by the NanoDrop 2000c Spectrophotometer at an absorbance level of 600 nm (Thermo Fisher Scientific Inc.) using cuvettes. LB media (100 μl) with or without rifampin hydrogel at a dilution of 1:105 were seeded on Mueller-Hinton agar plates containing oxacillin (6 μg/ml) for 48 hours, after which CFU numbers were counted using ImageJ software (33 (link)).
6
Kinetic Analysis of MabA Enzyme
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NADPH and acetoacetyl-CoA were purchased from Sigma-Aldrich. The reductase activity was determined by monitoring the decrease in the optical density at 340 nm corresponding to the oxidation of NADPH to NADP + . Kinetic constants for NADPH, acetoacetyl-CoA and -keto-octanoyl-CoA were determined with a fixed concentration of one component (1 mM for NADPH and 2 mM for acetoacetyl-CoA) and a range of concentrations of the second substrate. Assays were performed in a volume of 20 ml to determine the kinetic constants for acetoacetyl-CoA and -keto-octanoyl-CoA or in 80 ml for NADPH. Reaction mixtures consisted of 50 mM potassium phosphate buffer pH 7.0, the concentrations of substrates stated above and between 0.25 and 0.1 mM MabA MSMEG . The enzymatic reactions were initiated by the addition of acetoacetyl-CoA after incubation for 5 min at 25 C. The oxidation of NADPH was followed by removing 1.5 ml aliquots of the reaction mixture for analysis in a NanoDrop 2000c spectrophotometer (path length 1 mm; Thermo Scientific) to determine the kinetic constants of the -ketoacyl-CoAs and by continuous reading in an 80 ml quartz cuvette to determine the K m for NADPH in a NanoDrop 2000c spectrophotometer (path length 10 mm; Thermo Scientific). Experiments were performed in triplicate. Data were fitted using nonlinear least-squares regression with GraphPad Prism.
7
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted from FG and HG tissues by traditional phenol/chloroform extraction, using TRIzol Reagent (Invitrogen, Spain), and then purified and treated with DNase I using NucleoSpin® RNA Clean-up XS kit (Macherey-Nagel, Düren, Germany), according to guide instructions. Total RNA concentration, quality and integrity were evaluated using a NanoDrop 2000C Spectrophotometer (Fisher Scientific SL, Spain) and samples were stored at − 80 °C until complementary DNA (cDNA) synthesis.
cDNAwas synthetized from 1 μg of total RNA input using the qScript cDNA Synthesis Kit (Quanta BioScience), according to the manufacturer’s instructions, using the Applied Biosystems 2720 Thermal Cycler. The cycling conditions were 22 °C for 5 min, 42 °C for 30 min, and 85 °C for 5 min. Total RNA samples were stored at − 80 °C until gene expression was analysed.
cDNAwas synthetized from 1 μg of total RNA input using the qScript cDNA Synthesis Kit (Quanta BioScience), according to the manufacturer’s instructions, using the Applied Biosystems 2720 Thermal Cycler. The cycling conditions were 22 °C for 5 min, 42 °C for 30 min, and 85 °C for 5 min. Total RNA samples were stored at − 80 °C until gene expression was analysed.
8
RBM10 Isoform Characterization
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Full-length RBM10v1 and RBM10v2 amplicons were separated by agarose gel electrophoresis (note, no full-length RBM10v2 amplicon was detectable in the GLC20 cells) and cDNA was excised using a QIAquick gel extraction kit (Qiagen, Toronto, Canada). DNA quantity (absorbance at 260 nanometers (nm)) and purity (ratio of the absorbance at 260 and 280 nm) were determined using a NanoDrop 2000C spectrophotometer (Fisher Scientific, Ottawa, Canada), and the samples were sent for sequencing. Samples were sequenced, using the Sanger technique, by the MOBIX Lab - DNA Sequencing and Oligo Synthesis Facility (McMaster University, Hamilton, Canada). Internal primers (sequences available upon request) were generated by MOBIX. Bi-directional, overlapping sequence reads of ~600 bp were generated, as detailed in Figure 1 D.
9
Quantification of Gene Expression
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Adherent cells were pelleted and stored in RNAlater® (Life Technologies, Paisley, UK) at −80°C for future study. Messenger RNA was extracted from each cell pellet using the Isolate RNA Minikit (Bioline, London, UK) and adequate quantity and purity of extracted RNA were determined using a Nanodrop 2000c spectrophotometer (Fisher Scientific, Loughborough, UK). RNA samples were reverse transcribed to cDNA using a High Capacity RNA-to-cDNA Kit (Life Technologies, Paisley, UK) and real-time quantitative PCR carried out for each gene of interest using TaqMan™ gene expression assays (Life Technologies, Paisley, UK), as listed in Table 1 . All assays were conducted as described previously [16 (link)]. GAPDH was found to be stably expressed in a random selection of samples and was therefore used as the normalising gene in the current studies (data not shown). The 2−ΔCT formula was used to calculate the relative expression of mRNA of the genes of interest [17 (link)].
10
Intestinal RNA Extraction and cDNA Synthesis
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Total ribonucleic acid (RNA) was extracted from the anterior and posterior section of the intestine by traditional phenol/chloroform extraction, using the Trizol reagent (Invitrogen, Barcelona, Spain), and then purified and treated with DNase I using the NucleoSpin® RNA Clean-up XS kit (Macherey-Nagel, Düren, Germany), according to the manufacturer’s instructions. The concentration, quality and integrity of the total RNA were evaluated with a NanoDrop 2000C spectrophotometer (Fisher Scientific SL, Madrid, Spain). Only samples that obtained an absorbance ratio A260/280 between 1.8–2.0 and A260/230 greater than 2.0 were included in the analysis. The RNA samples were stored at –80 °C until the stage of complementary deoxyribonucleic acid synthesis (cDNA) to avoid RNA degradation.
Subsequently, the cDNA was synthesized from 1 μg of RNA using the qScript Flex cDNA kit (Quanta BioScience, Beverly, MA, USA), according to the manufacturer’s instructions and using the Applied Biosystems 2720 thermal cycler. The thermocycler conditions were 22 °C during 5 min, 42 °C for 30 min and 85 °C for 5 min. Once the cDNA was obtained, it was stored at –20 °C until the gene expression was analyzed.
Subsequently, the cDNA was synthesized from 1 μg of RNA using the qScript Flex cDNA kit (Quanta BioScience, Beverly, MA, USA), according to the manufacturer’s instructions and using the Applied Biosystems 2720 thermal cycler. The thermocycler conditions were 22 °C during 5 min, 42 °C for 30 min and 85 °C for 5 min. Once the cDNA was obtained, it was stored at –20 °C until the gene expression was analyzed.
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