Both strains CAPE95 and CAPE238 had their DNA isolated using the methods described by Rosado and Seldin (1993) (link). The bacterial cultures of each strain were centrifuged (10,000 × g for 15 min), concentrated in 5 mL of Tris-EDTA-NaCl buffer (pH = 8.0), treated with 500 μL of lysozyme (10 mg mL−1 for 1 h at 37°C) and 500 μL of 10% sodium dodecyl sulfate (10 min at 37°C). The DNA purification process was performed as described by Seldin and Dubnau (1985) (link). The concentration and purity of the DNA were determined using a Quibit™ 3.0 fluorometer (Thermo Fisher Scientific™, Waltham, MA, United States) and a Nanodrop spectrophotometer (Thermo Fisher Scientific™), respectively, guaranteeing a total genomic DNA concentration ≥ 200 ng, in a sample volume ≥ 20 μL and adopting the parameters of purity of A260/280 = 1.8–2.0. Furthermore, the DNA from both strains was sent to Novogene (SAC, United States). Paired-end libraries (2 × 150 bp) with a 350 bp insert size were constructed, and the whole genome was sequenced on an Illumina NovaSeq 6000 following the manufacturer’s recommendations.
Quibit 3.0 fluorometer
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The Qubit™ 3.0 fluorometer is a compact, high-performance instrument used for precise quantification of DNA, RNA, and protein samples. It utilizes fluorescent dyes to detect and measure the concentration of target analytes in a sample. The Qubit™ 3.0 provides accurate and reproducible results with a wide linear dynamic range.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Il 26, by Cusabio (1 mentions)
Cell death detection elisaplus, by Roche (1 mentions)
Dnaeasy kit, by Qiagen (1 mentions)
Thermal cycler, by Bio-Rad (1 mentions)
Beadbug homogeniser, by Benchmark Scientific (1 mentions)
Maxwell 16 instrument, by Promega (1 mentions)
4 protocols using quibit 3.0 fluorometer
1
Genomic DNA Isolation and Sequencing
2
Measuring Plasma NETs and Cytokines in COVID-19
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The presence of NETs in plasma was determined by measuring the concentration of double-stranded DNA (dsDNA) in a Quibit™ 3.0 Fluorometer (Thermo Fisher), and the levels of histone-complexed DNA (i.e., cell-free nucleosomes) using the Cell Death Detection ELISAPLUS (Roche). The protein concentrations of IL-6, IL-8, IL-26, and TNFα in human plasma were quantified using ELISA (IL-6, IL-8, and TNFα: R&D Systems; IL-26: Cusabio) according to the manufacturer’s instructions. Cytokine concentrations were measured in samples from all patients and controls, but the plasma concentration of dsDNA and cell-free nucleosomes could only be measured in 32 out of 49 patients from the COVID-19 group, and 26 out of 27 participants from the Control group. Results on the specific levels of dsDNA, cell-free nucleosomes, IL-6, IL-8, and TNFα were already available from a previous publication on this patient material (8 (link)). However, all data on IL-26, as well as all comparisons presented in the current study have not been published elsewhere.
3
Genomic Sequencing of Field-Collected Species
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For each target species, ten field-collected individuals were pooled to obtain enough DNA for genomic sequencing. DNA was extracted with a DNAeasy kit (Qiagen) and concentration quantified with a Quibit 3.0 fluorometer (ThermoFisher). Libraries and Illumina MiSeq sequencing were conducted at UC Berkeley qB3 Functional Genomics Lab for 4 million 2 × 300 base pair (bp), paired-end reads.
4
Microbial Community Analysis via RISA
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Ribosomal RNA (rRNA) intergenic spacer analysis (RISA) is a method of microbial community analysis to compare samples without culture-dependent bias. Samples were prepared as follows: turbid broths were incubated overnight and centrifuged at 1,400 g for 10 min at 20°C. The supernatant was discarded, and the pellet was re-suspended with 500 µL of TSB. Five hundred µL of 4 M guanidine isothiocyanate (UltraPure™, ThermoFisher Scientific, Newport, UK) was added and the suspension was mixed with Wizard™ infrared vortex mixer (Firsherbrand™, Fisher Scientific, Loughborough, UK) at 2,000 rpm. One mL of culture mix was added into the tubes containing 1 g of 0.1 mm diameter zirconia/silica beads (Thistle Scientific, Glasgow, UK) and suspensions were mixed in Bead Bug homogeniser (Benchmark Scientific, Cole-Parmer®, St Neots, UK) at 2,800 shaking speed for 2 min. DNA was amplified with Maxwell® 16 Instrument (Promega, Southampton, UK) and quantified by Quibit® 3.0 fluorometer (ThermoFisher Scientific, Newport, UK). The internal transcribed spacer (ITS) bacterial region between the 16S rRNA and 23S rRNA subunit genes was amplified with 1406F (TGYACACACCGCCCGT, Eurofins Genomics, Ebersberg, Germany) and 23SR (GGGTTBCCCCATTCRG, Eurofins Genomics, Ebersberg, Germany) primers by running RISA-PCR in thermal cycler (BIO-RAD, Watford, UK) [15] .
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