The genomes of the four PMQR-producing E. coli (LV46221, LV46743, LV36464, and LV27950) were characterized. Genomic DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen, Aarhus) and quantified using Qubit 1.0 Fluorometer (Invitrogen, Waltham). The Nextera XT DNA Sample Preparation Kit (Illumina, San Diego, CA) was used to prepare sequencing libraries from 1 ng of genomic DNA, according to the manufacturer's instructions. Paired-end sequencing of 150 bp reads was performed on a MiSeq (Illumina). Sequence reads were then trimmed and filtered according to quality criteria, and assembled de novo using CLC genomics workbench version 8.5.1 (QIAGEN, Aarhus). RAST (Rapid Annotation using Subsystem Technology) was used for subsystem annotation of the genomes (Aziz et al., 2012 (link); Overbeek et al., 2014 (link)).
Qubit 1.0 fluorometer
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany
The Qubit (1.0) Fluorometer is a compact, easy-to-use instrument designed for accurate quantification of nucleic acids and proteins. It utilizes fluorescence-based detection technology to provide precise measurements of sample concentrations.
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Lab products found in correlation
2100 bioanalyzer, by Agilent Technologies (26 mentions)
Truseq rna sample prep kit v2, by Illumina (17 mentions)
Qubit 1.0 fluorometer, by PerkinElmer (12 mentions)
Caliper gx labchip gx, by PerkinElmer (11 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (10 mentions)
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Rneasy plant mini kit, by Qiagen (6 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Turbo dnase, by Thermo Fisher Scientific (4 mentions)
Qubit rna br assay kit, by Thermo Fisher Scientific (4 mentions)
Agilent 2200 tapestation system, by Agilent Technologies (4 mentions)
Qubit dsdna br assay kit, by Thermo Fisher Scientific (4 mentions)
Hiseq 2000, by Illumina (4 mentions)
Truseq stranded mrna sample prep kit, by Illumina (4 mentions)
High pure pcr template preparation kit, by Roche (3 mentions)
Exp nbd104 native barcoding kit, by Oxford Nanopore (3 mentions)
Ligation sequencing kit sqk lsk109, by Oxford Nanopore (3 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
80 protocols using qubit 1.0 fluorometer
1
Genomic Characterization of PMQR-Producing E. coli
2
Quantitative mRNA Expression Analysis
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Samples were collected and lysed in Trizol (Invitrogen). The RNeasy mini kit (Qiagen) was used for mRNA extraction and iScriptTM cDNA synthesis kit (Biorad) for cDNA conversion. cDNA was quantified with Qubit® ssDNA Assay Kit on Qubit® 1.0 Fluorometer (Invitrogen). RT-qPCR was performed in triplicate samples on a LightCycler® 480 System (Roche) using iTaq Universal SYBR GREEN supermix (Biorad). The expression of genes was normalized to Gapdh. The relative gene expression was compared by one-way analysis of variance (ANOVA) followed by Tukey post-test using IBM® SPSS® Statistics Software.
3
Genotyping by Sequencing of Hybrid Families
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Whole genomic DNA was extracted from caudal fin clips for the four parents of the two crosses and from tail clips of 150 offspring of each family, using the commercial Thermo Scientific KingFisher Flex Cell and Tissue DNA kit. Individual DNA concentration was quantified using both a NanoDrop ND-8000 spectrophotometer (Thermo Fisher Scientific) and Qubit 1.0 Fluorometer (Invitrogen, Thermo Fisher Scientific). Double digest RAD sequencing library preparation was performed following Leitwein et al. (2016) (link). Briefly, DNA was digested with two restriction enzymes (EcoRI-HF and MspI) and the digested genomic DNA was then ligated to adapters and barcodes. Ligated samples with unique barcodes were pooled in equimolar proportions and CleanPCR beads (GC Biotech, Alphen aan den Rijn, The Netherlands) were used to select fragments sized between 200 and 700 bp. The size selected fragments were amplified by PCR and tagged with a unique index specifically designed for Illumina sequencing (Peterson et al. 2012 (link)). For progenies, PCR products were pooled in equimolar ratios into four pools. Each pool was sequenced in a single Illumina HiSequation 2500 (i.e., 75 F1 progenies per lane), producing 125-bp paired-end reads. The four parents were sequenced together in a single lane in order to obtain a higher coverage depth.
4
Sequencing of Piwi-Deficient Genomes
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DNA was extracted from ∼100 males from each wild-type and from thePiwi KD lines using the Qiagen DNeasy Blood&Tissue kit. The genomic DNA quality and quantity were evaluated using a NanoDrop™ One UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and a Qubit® 1.0 Fluorometer (Invitrogen, Carlsbad, CA, USA), respectively. Three micrograms of DNA were repaired using the NEBNext FFPE DNA Repair Mix (NEB M6630). End repair and dA-tailing were performed using the NEBNext End repair/dA-tailing Module (E7546, NEB). Ligation was then performed with the Ligation Sequencing Kit 1D (SQK-LSK108, ONT, for G0, and SQK-LSK109 ONT for wild type strains, G73 and G0-F100 samples). MinION sequencing was performed according to the manufacturer’s guidelines using R9.4.1 flow cells (FLO-MIN106, ONT) and a Nanopore MinIon Mk1b sequencer (ONT) controlled by the ONT MinKNOW software (version 18.3.1 for G0, version 19.05.0.0 for isogenic wild-type strains, and version 19.10.1 for the G73 and G0-F100 samples). Base calling was performed after sequencing using Albacore (version 2.3.3) for G0, and the GPU-enabled guppy basecaller in high accuracy mode for isogenic wild-type strains (version 3.1.5), G73 (version 3.3.3) and G0-F100 samples (version 3.4.4).
5
Comprehensive DNA Extraction Protocol
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Tissue samples were homogenized in Tissue Lysis Buffer using the MagNA Lyser instrument with the MagNA Lyser Green Beads Tubes (Roche Diagnostics GmbH, Manheim, Germany). Samples were digested with 4 mg/µL proteinase K (Roche Diagnostics GmbH) at 56 °C for 2 h and 1 h in the case of tissue and buffy coat samples, respectively. Genomic DNA was isolated using the High Pure PCR Template Preparation Kit (Roche Diagnostics GmbH) according to the manufacturer’s instructions. The RNA content of the samples was eliminated with the RNase A/T1 Mix (2 mg/mL of RNase A and 5000 U/mL of RNase T1, ThermoFisher Scientific, Vilnius, Lithuania) for 1 h at 37 °C. Genomic DNA was eluted in 100 µL RNase- and DNase-free water and stored at −20 °C until use. The concentration of dsDNA was determined using a Qubit 1.0 fluorometer with the Qubit dsDNA HS Assay Kit (Invitrogen, Waltham, MA, USA).
CfDNA was isolated with the Quick-cfDNA Serum and Plasma Kit (Zymo Research Corp, Irvine, CA, USA) from 3–5 mL plasma per patient. CfDNA was quality assessed by BioAnalyzer 2100 microcapillary electrophoresis system (Agilent Technologies, Santa Clara, CA, USA) and was quantified by the HS dsDNA Assay Kit with a Qubit 1.0 instrument (Invitrogen).
CfDNA was isolated with the Quick-cfDNA Serum and Plasma Kit (Zymo Research Corp, Irvine, CA, USA) from 3–5 mL plasma per patient. CfDNA was quality assessed by BioAnalyzer 2100 microcapillary electrophoresis system (Agilent Technologies, Santa Clara, CA, USA) and was quantified by the HS dsDNA Assay Kit with a Qubit 1.0 instrument (Invitrogen).
6
Total RNA Extraction from Frozen Tissues
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Total RNA was extracted with the use of the NucleoSpin RNA kit (Macherey-Nagel, Germany) after passing the liquid N2-snap frozen tissues through special filter columns (shredders) in order to homogenize them and to reduce viscosity. DNA was removed by an in-column recombinant DNase treatment. Total RNA was eluted in RNase-fee water and stored at −80 °C until further use. The absolute measurement of RNA concentration was determined by the Quant-iT RNA Assay kit in the Qubit 1.0 fluorometer (Life Technologies Invitrogen, USA) that employs a dye specific for RNA and not for DNA.
7
DNA Extraction from T Cells
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DNA was extracted from flow cytometry-sorted T cells using the Qiagen AllPrep DNA/RNA Mini Kit (Qiagen) according to the manufacturer’s instructions. Quantification of the extracted DNA was done employing a Qubit 1.0 Fluorometer (Invitrogen, Carlsbad, CA, USA).
8
Differential Expression Analysis of M. ulcerans
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(i) Ribosomal RNA (rRNA) removal: rRNA was depleted from the mycobacterial total RNA preparations with the RiboZero Epidemiology Illumina kit, which removes eukaryote and prokaryote rRNA in a single step.
(ii) Preparation of RNA-seq libraries: The RNA-seq libraries were prepared with the TruSeq Stranded Total RNA LT Sample Prep kit (Illumina). The quality of all libraries was checked with the DNA-1000 kit (Agilent) on a 2100 Bioanalyzer and quantification was performed with Quant-It assays on a Qubit 1.0 fluorometer (Invitrogen).
(iii) RNA-seq: Clusters were generated for the resulting libraries, with Illumina HiSeq SR Cluster Kit v4 reagents. Sequencing was performed with the Illumina HiSeq 2500 system and HiSeq SBS kit v4 reagents. Runs were carried out over 65 cycles, including seven indexing cycles, to obtain 65-bp single-end reads. Sequencing data were processed with Illumina Pipeline software (Casava version 1.9). All 65-bp reads were aligned against the complete genome and plasmid sequences of M. ulcerans (Agy99 strain) obtained from the Burulist database with Bowtie software, and against the mouse genome (GRCm38) obtained from the Ensembl database with STAR software. Data were normalized and analyzed in R, with the Bioconductor packages.
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9
Amplicon Capture Library Preparation
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For the potato blight and pigeon samples used in the amplicon capture experiment, additional primers sets that amplify a 100 bp amplicon within the corresponding 250 bp marker were developed for use as bait (Table S2 ). The amplification conditions were the same as described above for the 250 bp markers. The 100-bp markers for the amplicon capture experiment were converted to bait without fragmentation.
The unmodified 250-bp amplicons and the amplicons with M13 flanking regions were purified using agarose gel purifications to remove residual primers. The four samples were then mixed equimolar according to DNA concentration as measured on a Qubit 1.0 Fluorometer (Invitrogen, Carlsbad, CA). The samples were converted to Illumina-compatible libraries using a NEBNext DNA Sample Prep Master Mix Set 2 kit (New England Biolabs, E6070L) without any fragmentation. The libraries were amplified using Phusion Flash High-Fidelity PCR Master Mix with 16 PCR cycles.
The unmodified 250-bp amplicons and the amplicons with M13 flanking regions were purified using agarose gel purifications to remove residual primers. The four samples were then mixed equimolar according to DNA concentration as measured on a Qubit 1.0 Fluorometer (Invitrogen, Carlsbad, CA). The samples were converted to Illumina-compatible libraries using a NEBNext DNA Sample Prep Master Mix Set 2 kit (New England Biolabs, E6070L) without any fragmentation. The libraries were amplified using Phusion Flash High-Fidelity PCR Master Mix with 16 PCR cycles.
10
Genomic DNA Extraction from Cells
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Genomic DNA (gDNA) was isolated using the GenElute Bacteria Genomic DNA Kit (Sigma- Aldrich, Dorset, UK) according to the manufacturer’s instructions and eluted with 200 μl of elution buffer. gDNA was purified from THP-1 cells and human challenge model samples using the QIAmp DNA mini and blood extraction kit (QIAgen, Manchester, UK) as per the manufacturer’s protocol. gDNA was quantified using a Qubit 1.0 fluorometer (Invitrogen, Loughborough, UK) according to the manufacturer’s instructions.
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