Quant it picogreen dsdna reagent kit

Amplicon libraries and sequencing was performed according to previously published protocols49 (link). Briefly, the V4 region of the 16S rRNA gene was PCR amplified from each DNA sample in triplicate using the 515f/806r primer pair described previously49 (link). Pooled amplicons from the triplicate reactions were purified using AMPure XP purification (Agencourt, Massachusetts, USA) according to the manufacturer’s instructions and eluted in 25 μl 1× low TE (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) and subsequently quantified by Quant-iT PicoGreen dsDNA reagent kit (Invitrogen, New York, USA) using a Victor3 Multilabel Counter (Perkin Elmer, Waltham, USA). Amplicons were mixed in equimolar concentrations, to ensure equal representation of each sample, and sequenced on an Illumina MiSeq instrument.

DNA from frozen tissues was extracted using the QIAamp DNA Mini Kit (Qiagen, Germantown, MD, USA). FFPE tissue DNA was extracted using GeneJet FFPE DNA Purification Kit and RecoverAll™ Total Nucleic Acid Isolation (Invitrogen, Thermo Fisher Scientific, Carslbad, CA, USA), following the manufacturer’s instructions, with overnight lysis instead of the suggested 1–2 h for FFPE tissue. Germline DNA was purified from whole blood samples or buffy coat using the Wizard^® Genomic DNA Purification Kit (Promega, Madison, WI, USA), according to manufacturer’s protocol.
Purified DNA was quantified using the Qubit(™) dsDNA HS Assay and Quant-IT(™) Picogreen^® dsDNA Reagent Kit (Invitrogen, Thermo Fisher Scientific, Carslbad, CA, USA). The purity of DNA was assessed by measuring the 260/280 nm absorbance ratio. For FFPE samples, fragment length and degradation were assessed using the HS Genomic DNA Analysis Kit (DNF-488) in a Fragment Analyzer (Agilent, formerly Advanced Analytical). DNA ranged from >1000 bp to 200 bp. Samples with <200 bp are not recommended for processing with the TumorSec workflow.

Detection of DNA in 2 μL of citrated plasma samples was performed using the Quant-iT™ PicoGreen^® dsDNA reagent kit and Lambda DNA standard (Invitrogen, CA, USA, cat# P7589) according to the manufacturer’s instructions. Briefly, 98 μL of TE buffer were aliquoted in 96-well plate, 2 μL of plasma were added followed by 100 μL of PicoGreen^® dsDNA reagent. Fluorescence was measured on a POLARstar Omega microplate reader (BMG Labtech, Germany) at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. Plasma DNA concentration was determined by plotting fluorescence on the Lambda DNA standard curve.

The cell-laden hydrogels were incubated at 37 °C and under 5% CO₂ conditions for 1, 7, 14 and 28 days for the double-stranded DNA (dsDNA) content analysis. On specific days, the culture medium was removed, and the samples were washed with PBS 3 times. The weight of the samples was recorded, and the samples were stored at −60 °C until the entire culture study was finished. The study was conducted using a Quant-iT™ PicoGreen dsDNA Reagent kit (Invitrogen, Carlsbad, CA, USA) by following the manufacturer’s instructions. Briefly, the cell-laden hydrogels were homogenized in a 1X TE buffer with a glass tissue grinder (Wheaton, Primary, MN, USA), and further cell lysis was carried out using a freeze and thaw method. The sample solutions and Quanti-iT™ PicoGreen reagent were mixed at a ratio of 1:1 and placed in a black 96-well plate (Thermo Fisher Scientific, Waltham, MA, USA). The process was performed with protection from light. Standard curves were made with concentrations ranging from 0 to 2 ug/mL. The quantification was carried out using a microplate reader (EMax, Molecular Device, Sunnyvale, CA, USA) with the fluorescence intensities at an excitation wavelength of 485/20 nm and an emission wavelength of 528/20 nm.

Genomic DNA was extracted from EDTA blood or mouthwash samples using the FlexiGene DNA kit (Qiagen GmbH, Hilden, Germany) and quantified using Quant‐iT Pico Green dsDNA reagent kit (Invitrogen/Life Technologies, Darmstadt, Germany). Of 492 selected SNPs, 447 passed quality control after genotyping (for 45, success rate was below 95%, seven were not in Hardy–Weinberg equilibrium (HWE) and were selected to be genotyped on the customized GoldenGate assay (Illumina, San Diego, CA) 23. The iPLEX assay (Sequenom, Hamburg, Germany) for the MassArray system was used to genotype five SNPs that failed genotyping on the Illumina GoldenGate platform 24. Quality of genotyping was high with concordance of duplicates from Centre d'Etude du Polymorphisme Humain (Paris, France) and control samples above 98%. The two selected non‐SNP polymorphisms in the TYMS gene were genotyped using fragment analysis and single‐strand conformation polymorphism in the laboratory of Dr. Ulrich at the National Center for Tumor Diseases in Heidelberg, Germany.

Genomic DNA was extracted from 48–72 h cultures grown in BHIgg broth. The collected cells were treated with 1 mg/mL lysozyme in DNA extraction buffer (200 mM Tris-HCl, 25 mM NaCl, 25 mM EDTA, pH8.5) at 37°C for 30 min. The lysed cells were further treated by 1% SDS at 70°C for 10 min. Following the cell lysis, genomic DNA was extracted using a phenol-chloroform extraction method. A 4-μg of genomic DNA was then treated using a Nextera DNA Sample Prep kit (Illumina, Tokyo, Japan) according to the manufacturer’s instructions. The quality of the libraries was confirmed by Agilent 2100 Bioanalyzer using a High Sensitivity DNA kit (Agilent Technologies, Santa Clara, CA, USA), while a Quant-iT PicoGreen dsDNA Reagent kit (Invitrogen, Carksbad, CA, USA) was used to quantify the libraries. The denatured library mixture was sequenced using a Miseq sequencer (Illumina) according to the manufacturer’s instructions, using a Miseq Reagent Kit v2 (300 cycle; Illumina). The raw sequence data were assembled using the CLC Genomics Workbench (CLC Bio, Tokyo, Japan) and annotated by the Microbial Genome Annotation Pipeline (MiGAP; http://www.migap.org). The genome sequence was deposited in the DDBJ Sequence Read Archive (DRA) under the accession numbers BBXO01000001−BBXO01002032.