Quick dna hmw magbead kit

Wild B. violaceus colonies were collected from Morro Bay, CA (CDFW permit GM-190280002-19028) and subsequently cleared of visible detritus with forceps and brush. Colonies were cultivated and starved in filtered (0.22 $\mu$ m) seawater at 10 ${}^{\circ }$ C for approximately two days to reduce potential microbial contamination from the gut. Approximately, 100 $\mu$ L of blood was extracted from a colony using a 1 mL needle and syringe. DNA for Illumina sequencing was isolated using a Qiagen DNeasy Blood & Tissue Kit according to manufacturer instructions. Initial ONT sequencing using the same DNA used for Illumina sequencing produced low quality sequencing data (data not shown). From another colony, enriched blood was acquired by macerating 1 g of colony with a syringe plunger against a 40 $\mu$ m sieve (Falcon™ Cell Strainer—Fisher Science) as previously described (Rosental et al. 2018 (link)). Over 5 $\mu$ g of DNA was extracted per 200 $\mu$ L enriched blood using the Zymo Quick-DNA HMW MagBead Kit. Manufacturer’s instructions were followed except for increasing the volume of magnetic beads to 50 $\mu$ L total. Samples were visually assessed via gel electrophoresis and quantified with Qubit Fluorometric Quantitation (ThermoFisher, Inc.).

Singular bacteria colonies were picked from CSM agar (pH 6.5) and incubated in 20 mL Tryptic Soy Broth medium in 150 mL baffled shaking flasks at 120 rpm and 28°C overnight. HMW gDNA was extracted according to the instructions of the Quick‐DNA HMW MagBead Kit (Zymo Research).
To assess the quantity and purity of the obtained DNA, 260/280 nm absorption ratios and concentrations were measured with a photometer (Nano Photometer NP80; IMPLEN) and a Qubit 4 fluorometer with the Qubit 1X dsDNA HS Assay‐Kit (Thermo Fisher Scientific). To confirm the high molarity of the gDNA, fragment sizes were analyzed with a Femto Pulse capillary electrophoresis instrument (Agilent Technologies).
When samples passed the quality control, shearing of 8 µg gDNA in 150 µL Elution Buffer was conducted with g‐TUBEs (Covaris), utilizing 1700g in a tabletop centrifuge. This yielded DNA fragments with a size of ca. 12 kbp, as confirmed with Femto Pulse. Subsequently, HiFi libraries were prepared according to the SMRTbell prep kit 3.0 manual, fusing barcoded adapters to the samples (Pacific Biosciences). Libraries were stored at −20°C until the day of sequencing, where primers and the polymerase bound the samples with the Sequel II Binding Kit 3.2 (Pacific Biosciences), closely following the manufacturer's recommendations.

Ten milliliters of thawed food sample primary enrichments were centrifuged for 5 min at 6000 × g at 4°C and the pellet was resuspended in 400 μL of DNA/RNA Shield (Cedarlane). DNA was extracted using the Quick-DNA HMW MagBead Kit with RNAse A treatment according to the manufacturer’s protocol (Zymo Research Corp.). DNA was quantified using a Qubit fluorometer (Thermo Fisher Scientific). Microbial DNA was enriched using the NEBNext Microbiome DNA Enrichment kit (New England Biolabs) according to the manufacturer’s instructions and the Collect Enriched Microbial DNA protocol. Enriched DNA samples were purified using 1.8x AMPure XP beads following the AMPure XP Bead Cleanup protocol (Beckman Coulter).

Genomic DNA of strain ICN-92133^T was extracted using Quick-DNA HMW MagBead kit (Zymo Research Corporation, Irvine, CA, USA) with lysozyme chloride (Sigma-Aldrich, St. Louis, MO, USA) and quantified by QuantiFluor dsDNA System method (Promega, Madison, WI, USA) using Victor Nivo Multimode Microplate Reader (PerkinElmer, Waltham, MA, USA). Whole genome sequencing was performed using a PacBio Sequel (Pacific Biosciences, Menlo Park, CA, USA) and a NovaSeq 6000 system (Illumina, San Diego, CA, USA) by Macrogen Corporation (Seoul, South Korea). PacBio long-read sequences from approximately 12 kb SMRTbell templates were assembled de novo to construct genome sequences. They were then corrected using Illumina sequencing data from paired-end (2 × 150 bp) sequencing to obtain high quality. In this study, Microbial Assembly Application was applied for assembly¹⁸ , and the genome was annotated using the PATRIC RAST tool kit (RASTtk)^{19 (link)} in the BV-BRC server^{20 (link)}. The circular genome was constructed in a CGView tool^{21 (link)}. The virulence factor database (VFDB)^{22 (link)} and the Comprehensive Antibiotic Resistance Database (CARD)^{23 (link)} were used to predict potential virulence factors and antimicrobial resistance genes.

All chemicals were supplied from Sigma-Aldrich (Darmstadt, Germany), and general consumables were obtained from VWR (Darmstadt, Germany). All necessary buffers and enzymes for next-generation genome sequencing were shipped from Pacific Biosciences (Menlo Park, CA, USA). High molecular weight DNA was extracted with the Quick-DNA™ HMW MagBead Kit from Zymo Research (Freiburg, Germany). HMW gDNA shearing was conducted with g-TUBEs (Covaris, Woburn, MA, USA).