Qubit dsdna broad range assay

ATAC-Seq was performed following the protocol described by Buenrostro and colleagues [40 ]. Propagation and embryoid body (EB)-based differentiation of the doxycycline-inducible Pax3 ES cell line was performed as previously described [41 ]. Pax3 induction was achieved by adding doxycycline (final concentration of 1 μg/ml) in 3-day EB cultures. Fifty thousand freshly sorted PDGFRa+FLK1− cells from cultures differentiated for 4 days (non-induced and 1-day Pax3-induced cells) were washed with 200 μl of cold PBS then resuspended in 100 μl of cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% IGEPAL CA-630), spun at 500 g for 10 min at 4 °C, and resuspended in 50 μl of the transposition reaction mix. Transposition occurred at 37 °C for 30 min, after which transposed DNA was purified using a Qiagen MinElute Kit and eluted in 12 μl Elution Buffer. Transposed DNA was quantified using qPCR, followed by the final PCR amplification using Illumina-compatible adapter-barcodes (using the forward indexing primers and reverse indexing primers described above). Three independent libraries were generated for both non-induced and Pax3-induced cells. Libraries were quantified using a Qubit dsDNA broad-range assay (Thermo Fisher Scientific), fragment sizes were assessed using an Agilent Bioanalyzer High Sensitivity assay, and libraries were normalized to 2 nM for sequencing.

Genomic DNA for WGS was extracted from mutant embryonic liver samples using the QIAGEN DNeasy Blood and Tissue Kit (#69504) according to the manufacturer’s instructions, but with some modifications. Approximately 10 mg of embryonic liver was used, and was snipped into very small pieces in individual plastic weigh boats with fine dissecting scissors. Lysis buffer was used to collect all of the tissue from the weigh boat. Samples were digested with the Proteinase K provided in the kit (20 mg/ml) for 1 hr, with vortexing every 10 min. RNA was digested with 8 μl of RNase A (QIAGEN #19101, 100 mg/ml at 7000 units/ml) for 2 min. Two elutions were conducted into two separate microfuge tubes, with elution buffer incubated on the column for 5 min before centrifugation. DNA was tested at SCRI for purity and quality using a Nanodrop spectrophotometer and the Qubit dsDNA Broad Range Assay (#Q32850; Thermo Fisher Scientific). DNA samples were shipped to HudsonAlpha Genomic Services Laboratory (Huntsville, AL) or NovoGene (Chula Vista, CA), for WGS.

EBUS-TBNA needle aspirates were homogenized using 1.4 mm ceramic beads (Sapphire Biosciences, Sydney, Australia) in a Precellys 24 device (Bertin Technologies, Montigny-le-Bretonneux, France). DNA and RNA were extracted using AllPrep^® DNA/RNA (Qiagen, Clayton, Australia). DNA was extracted from whole blood using the QIAamp^® DNA Blood Kit (Qiagen). DNA samples were quantified using the Qubit^® dsDNA broad-range Assay (Thermo Fisher Scientific Australia, Scoresby, Australia), and the DNA and RNA integrities of the tumor samples were evaluated using the 4200 TapeStation System (Agilent Technologies Australia, Mulgrave, Australia).

Irrigation water and fresh produce samples from spinach production systems were collected and ESBL-producing Enterobacterales were isolated as described (Richter et al., 2020 (link)). A selection of 19 isolates were further characterized (Table 1). The genomic DNA of each isolate was extracted with the DNeasy PowerSoil kit (Qiagen, South Africa) according to the manufacturer’s instructions. Following gDNA extraction, the concentrations were determined using the Qubit dsDNA Broad Range Assay and a Qubit 2.0 fluorometer (Life Technologies, Johannesburg) and quantification was determined on a Nanodrop 2000 (ThermoScientific, Johannesburg).

Genomic DNA from tissue specimen and corresponding blood samples were extract-ed with the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). DNA quantitation was assessed by the Qubit dsDNA Broad-Range Assay (Life Technologies, Darmstadt, Germany).