Briefly, genomic DNA was extracted from formalin-fixed, paraffin-embedded tumor tissues and whole blood according to the manufacturer’s standard protocol (Institute of Pathology, Southwest Hospital, Chongqing, People’s Republic of China). The concentration of the DNA samples was measured with the dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA) using a Qubit Fluorometer to ensure that genomic DNA was greater than 30 ng. Then, DNA shearing was performed using Covaris M220, followed by end repair, phosphorylation, adaptor ligation and polymerase chain reaction (PCR) amplification. The purified pre-enrichment library was hybridized to an OncoScreenPlusTM (Burning Rock, Guangzhou, China) panel covering 520 human cancer-related genes as well as more than 9000 single-nucleotide polymorphisms (SNPs) located throughout the genome, followed by hybrid selection with magnetic beads, and PCR amplification. The quality and size distribution of the libraries were assessed by a dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA) using a Qubit Fluorometer and a High Sensitivity D1000 ScreenTape kit using 4200 TapeStation (Agilent Technologies, CA, USA). Indexed samples were then sequenced on a NextSeq sequencer (Illumina, San Diego, CA) with paired-end reads (read length, 150 bp) and an average sequencing depth of 1000× for tissue samples and 200× for whole blood samples.
Dsdna hs assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, Canada
The DsDNA HS Assay Kit is a fluorescence-based reagent system designed to quantify double-stranded DNA (dsDNA) in solution. The kit utilizes a dye that selectively binds to dsDNA, allowing for the sensitive detection and measurement of DNA concentration.
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Lab products found in correlation
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (55 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (38 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (30 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (29 mentions)
Dneasy blood tissue kit, by Qiagen (28 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (27 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (18 mentions)
2100 bioanalyzer, by Agilent Technologies (18 mentions)
Dneasy blood and tissue kit, by Qiagen (14 mentions)
Qubit 3, by Thermo Fisher Scientific (14 mentions)
Kapa hyper prep kit, by Roche (14 mentions)
Qiaamp circulating nucleic acid kit, by Qiagen (14 mentions)
High sensitivity dna kit, by Agilent Technologies (10 mentions)
Agencourt ampure xp bead, by Beckman Coulter (10 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (9 mentions)
Miseq platform, by Illumina (9 mentions)
Qubit 2.0 fluorimeter, by Thermo Fisher Scientific (9 mentions)
Dneasy plant mini kit, by Qiagen (8 mentions)
Ampure xp bead, by Beckman Coulter (8 mentions)
Dneasy powersoil kit, by Qiagen (8 mentions)
284 protocols using dsdna hs assay kit
1
Comprehensive Genomic Profiling of Tumor Tissue
2
Comprehensive Genomic Profiling of Tumor Tissue
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Briefly, genomic DNA was extracted from formalin-fixed, paraffin-embedded tumor tissues and whole blood according to the manufacturer’s standard protocol (Institute of Pathology, Southwest Hospital, Chongqing, People’s Republic of China). The concentration of the DNA samples was measured with the dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA) using a Qubit Fluorometer to ensure that genomic DNA was greater than 30 ng. Then, DNA shearing was performed using Covaris M220, followed by end repair, phosphorylation, adaptor ligation and polymerase chain reaction (PCR) amplification. The purified pre-enrichment library was hybridized to an OncoScreenPlusTM (Burning Rock, Guangzhou, China) panel covering 520 human cancer-related genes as well as more than 9000 single-nucleotide polymorphisms (SNPs) located throughout the genome, followed by hybrid selection with magnetic beads, and PCR amplification. The quality and size distribution of the libraries were assessed by a dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA) using a Qubit Fluorometer and a High Sensitivity D1000 ScreenTape kit using 4200 TapeStation (Agilent Technologies, CA, USA). Indexed samples were then sequenced on a NextSeq sequencer (Illumina, San Diego, CA) with paired-end reads (read length, 150 bp) and an average sequencing depth of 1000× for tissue samples and 200× for whole blood samples.
3
Extracting Nucleic Acids from FFPE Tissues
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For FFPE tissues, tumor-rich areas (>30-50% of neoplastic cells) were microdissected from three to six 4-μm unstained histologic sections under stereomicroscopic visualization with an Olympus SZ61 microscope (Olympus, Hamburg, Germany). Total nucleic acids were isolated from each target with the DNeasy Blood and Tissue kit on the automated QIAcube (Qiagen, Valencia, CA) instrument according to the manufacturer's instructions. Extracted DNA and RNA were quantitated on the Qubit 2.0 Fluorometer using the dsDNA HS Assay Kit and the RNA HS Assay Kit (Invitrogen, Carlsbad, CA).
4
Ion Torrent Whole Genome Sequencing
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Library construction was conducted as per the Ion plus fragment library kit (Invitrogen Cat. No 4471252) for whole genome libraries. Total genomic DNA input was 100 ng which was fragmented using Ion shearTM enzyme mix II enzyme with an average of 400 bp DNA fragment sizes. The fragmented genomic library was cleaned using Agencourt Ampure XP Reagent (Beckman Coulter). The fragmented DNA was quantified using the Qubit DSDNA HsAssay Kit with the Qubit Fluorometer (Invitrogen). Purified DNA fragments were ligated with adapters specific to cleavage site of endonuclease enzyme, followed by the size selection of genomic library using E-gel 2% in order to get fragment size of 350–400 bp. The library was amplified using 10 cycles of PCR for enrichment of adapter ligated fragments and purified with Agencourt Ampure XP Reagent (Beckman Coulter). Template preparation for sequencing was conducted according to the OneTouch Ion™ Template Kit (Life Technologies). The selected PCR products were again used for emulsion PCR, followed by positive bead recovery. Ion Torrent sequencing was conducted using the Ion PGMTM 400 Sequencing Kit (Life Technologies) on an Ion Torrent Personal Genome Machine (PGMTM, Life Technologies) using a 318V2-chip (Ion 318TM chip, Life Technologies).
5
RNA Isolation and Sequencing for Fungal Gene Prediction
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We did not have enough mycelium from O. camponoti-rufipedis to generate an RNA sample to aid in gene prediction. For the other strains, enough mycelium was harvested so 1 cm2 mycelium could be used for RNA isolation. Total RNA was extracted as previously reported39 (link). A Bioanalyzer System with the RNA 6000 Nano Kit (Agilent Technologies) and a Qubit with the RNA BR Assay Kit (Invitrogen) were used to verify sample quality and quantity. mRNA-Seq libraries were constructed with a NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs). Quality and quantity of the resulting cDNA libraries were verified with a Qubit using the ds DNA HS Assay Kit (Invitrogen) and a Bioanalyzer System with the High Sensitivity DNA Analysis Kit (Agilent Technologies). Samples were sequenced on a HiSeq. 1500 (Illumina) using 100 bp paired-end sequencing in rapid-run mode.
6
Viral DNA Extraction and Sequencing
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For initial PCR-based analysis of PolB, we extracted viral DNA using Wizard columns (Promega, USA). For genome sequencing, 5 ml of each of the three viral stocks was filtered onto 0.1 µm, 45 mm Supor filters (Pall Scientific, USA) and flash-frozen in liquid nitrogen. This material was extracted using a modified CTAB protocol [15 (link), 43 (link)] designed to maximise the capture of unfragmented DNA molecules. DNA was quantified using a Qubit with the dsDNA HS Assay kit (Invitrogen, USA).
7
Exploring DNA Methylation via epiGBS Sequencing
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We used an existing protocol called epiGBS [30 , 82 (link)], a reduced representation bisulfite-sequencing method for cost-effective exploration of DNA methylation and genetic variation designed for multiplexed high-throughput sequencing to maximize sample size while losing loci. epiGBS sequencing was performed with the snails exposed to the DNMTi that showed the most significant changes in the global 5mC%. Eight samples per treatment were sequenced from control group, Flv1-treated, and from the progeny of control and the Flv1-treated group. 32 DNA isolated samples were quantified with Qubit fluorometer with the dsDNA HS Assay Kit (Invitrogen). The concentration was homogenized in all samples to 10 ng/µL in a total volume of 35 µL. epiGBS library preparation was applied as described in the step-by-step most recent protocol [30 ]. Paired-end sequencing (2 × 150 bp) using an Illumina NextSeq™550 instrument at the Bio-Environment NGS Platform at the University of Perpignan.
8
RNA-seq Library Preparation from OM Cells
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Total RNA of OM cells cultured at passages 4–5 was extracted using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions with a minor adjustment of addition of β-mercaptoethanol to RLT Plus reagent. Agilent 2100 Bioanalyzer with the RNA 6000 Pico kit was used to evaluate integrity of the isolated RNA (Agilent, Santa Clara, CA, USA). Prior to RNA-seq library preparation, rRNA was depleted from 100 ng of total RNA input with QIAseq Fast Select RNA Removal Kit (Qiagen) following the manufacturer’s instructions. RNA libraries were prepared using QIAseq Stranded Total RNA Library Kit (Qiagen), according to the manufacturer’s instructions. Concentrations of the amplified libraries were measured with Qubit fluorometer and dsDNA HS assay kit (Invitrogen, Waltham, MA, USA). Library size distribution was visualized with Agilent 5200 Fragment Analyzer and HS NGS Fragment Kit (1–6000 bp). Libraries were pooled and subsequently sequenced on Illumina NovaSeq 6000 platform using S1 Reagent Kit (Illumina, San Diego, CA, USA). The 2 × 100 bp paired-end sequencing resulted in approximately 50 million reads per sample.
9
Bacterial and Plant DNA Extraction
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Total bacterial DNA was extracted from 1 mL of bacterial cultures of Xcc NCPPB 3234 using Gentra Puregene Yeast/Bact. Kit (Qiagen, Venlo, The Netherlands). The DNA concentration was evaluated by Qubit (dsDNA HS Assay kit, Invitrogen, Waltham, MA, USA). Plant DNA extraction was performed following the manufacturers’ instructions of the commercial kits, i.e., DNeasy Plant Mini kit (Qiagen, Venlo, The Netherlands), DNeasy Mericon TM Food Kit (Qiagen, Venlo, The Netherlands), and QuickPick™ SML Plant DNA kit (QRET Technologies Ltd., Turku, Finland), manual version, from 500 mL of plant extract. The Ctab DEM was performed as described for Xylella fastidiosa [16 ]. The DNA was stored ≤−15 °C until analysis.
10
ASFV LAMP Assay with Exogenous IAC
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A dsDNA gBlock Gene Fragment (Integrated DNA Technologies Inc., Coralville, IA, USA) was synthesised as a positive control for ASFV and initial primer optimization. The gBlock was made by aligning the two outer LAMP primers (F3/B3) [36 (link)] (Table 1 ) to Malawi Lil-20/1 ASFV genome (GenBank accession AY261361), covering a 206 bp region of the TPII (Table 2 ). The gBlock was amplified using 1 µL of each F3 and B3 ASFV LAMP primer at 10 µM using PlatinumTM PCR SuperMix High Fidelity (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. The amplified product was purified using an Isolate II PCR and Gel Kit (Bioline, Taunton, MA, USA) and quantified using dsDNA HS Assay Kit on a QubitTM 2.0 (Invitrogen Carlsbad, CA, USA) fluorometer. A second gBlock was designed to be used as an exogenous internal amplification control (IAC). Briefly, the IAC gBlock targeted the same region as the ASFV gBlock apart from the addition of 8 nucleotides (Table 2 ) to elevate the annealing temperature. The IAC gBlock was prepared as outlined above.
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