Strains Z1723 and Z1767 were cultured from a single colony in LB medium to an OD600=0.7. 1 ml of cells/agarose plug were harvested (4000 g, 5 min) and intact chromosomes were extracted according to the Bionano Prep Cell Culture DNA Isolation Protocol (Bionano). Briefly, harvested cells were washed twice in Bionano Cell Buffer (Bionano). Washed cells were embedded in 2% low-melt agarose plugs and cells were lysed (1 h at 37 °C) with lysozyme enzyme (100 µl) using CHEF Bacterial Genomic DNA Plug Kit (Bio-Rad). DNA containing plugs were washed twice with nuclease free water then treated with Proteinase K (Qiagen) in Bionano Lysis Buffer according to the Bionano Prep Cell Culture DNA Isolation Protocol. All subsequent procedure steps (RNase treatment, DNA extraction, quantitation and labelling) and optical mapping on Bionano Irys platform were provided as a service by Earlham Institute (Norwich, UK). Structural variant analysis was provided by Bionano and structural variant maps visualized using Bionano access (Bionano).
Bionano prep cell culture dna isolation protocol
Manufactured by Bionano Genomics
The Bionano Prep Cell Culture DNA Isolation Protocol is a laboratory procedure designed to extract high molecular weight DNA from cell cultures. The protocol outlines the steps required to isolate DNA samples suitable for use with Bionano Genomics' platform technologies.
Automatically generated - may contain errors
Lab products found in correlation
Nuclei prep buffer, by Zymo Research (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Saphyr instrument, by Bionano Genomics (1 mentions)
Dle 1 enzyme, by Bionano Genomics (1 mentions)
Dl green, by Bionano Genomics (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Bionano access, by Bionano Genomics (1 mentions)
Irys platform, by Bionano Genomics (1 mentions)
Chef bacterial genomic dna plug kit, by Bio-Rad (1 mentions)
Proteinase k, by Qiagen (1 mentions)
Ligation sequencing kit sqk lsk109, by Oxford Nanopore (1 mentions)
R9.4.1 flow cell, by Oxford Nanopore (1 mentions)
Promethion, by Oxford Nanopore (1 mentions)
Novaseq, by Illumina (1 mentions)
4 protocols using bionano prep cell culture dna isolation protocol
1
Genomic DNA Extraction from Bacterial Cells
2
WI-38 Nuclei Optical Mapping
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WI-38 nuclei were isolated using isolation buffer from Nuclei Prep Buffer (Zymo Research). Isolated nuclei were embedded into agarose plugs and high molecular weight (HMW) genomic DNA was extracted according to the Bionano Prep Cell Culture DNA Isolation Protocol (Bionano Genomics). HMW DNA was labeled with one of three labeling assays: nicking enzymes Nb.BssS I (NEB) and Nt.BspQI (NEB) were used to label two 300 ng aliquots the HMW DNA according to the Prep Labeling - NLRS Protocol (Bionano Genomics Document 30024H), while a third aliquot (750 ng) of HMW DNA was labeled using the DLE-1 Enzyme assay (Bionano Genomics Document 30206A). Labeled DNA molecules detected using the Saphyr System optical mapper (Bionano Genomics).
3
Long-read DNA Extraction and Labeling
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Images of human DNA fragments (a total of 445 images, see one example in Fig. 1 ), were used to estimate the label detection likelihood parameters (Section 2.1.2). These images were captured using the Saphyr instrument and Saphyr chips (G1.2) (Bionano Genomics). The protocol for extraction and labeling of DNA is described in Nogin et al. (2023) (link). U2OS human cell line was cultured in Dulbecco’s modified Eagle medium with 10% fetal bovine serum (Gibco, Amarillo, TX), 2 mM l -glutamine, and 1% penicillin-streptomycin (10 000 U/ml; Gibco). The cells were incubated at 37°C with 5% CO2. DNA was extracted from cells using the Bionano Prep Cell Culture DNA Isolation Protocol (Bionano Genomics). One microgram of DNA was directly labeled using the Bionano Genomics DLS labeling kit, composed of a single enzymatic labeling reaction with DLE-1 enzyme. The reaction mixture contained 6 µl of 5× DLE-buffer, 2 µl of 20× DL-Green, 2 µl of DLE-1 enzyme (Bionano Genomics), and a total reaction volume of 30 µl. The reaction was incubated for 2 h at 37°C.
4
Tubeworm Genomics: Nanopore and Illumina Sequencing
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Tubeworms were collected with the Nanseimaru at Kagoshima Bay, Kagoshima, Japan (31_39.756ʹN, 130_48.050ʹE), kept in a tank, and immediately transferred to Kagoshima City Aquarium (25 June 2018). Chitinous tubes were removed, and then the obturacular region, vestimental region, and trunk region were cut into approximately 1 cm pieces. The male gonad with sperm, was isolated from male specimens. Samples were immediately stored at −80℃ until DNA and RNA extraction.
To obtain highly concentrated DNA with little contamination from symbionts, we isolated high-molecular-weight DNA from frozen male gonads, according to the Bionano Prep Cell Culture DNA Isolation Protocol (BioNano Genomics). This isolated, high-molecular weight DNA was used to construct a sequencing library with Ligation Sequencing Kit (SQK-LSK109; Oxford Nanopore Technologies) according to the manufacturer’s instructions. The library was sequenced with PromethION (Oxford Nanopore Technologies) using R9.4.1 flow cells (Oxford Nanopore Technologies). The same DNA was also sequenced using a NovaSeq (250 bp, paired-end) (Illumina).
To obtain highly concentrated DNA with little contamination from symbionts, we isolated high-molecular-weight DNA from frozen male gonads, according to the Bionano Prep Cell Culture DNA Isolation Protocol (BioNano Genomics). This isolated, high-molecular weight DNA was used to construct a sequencing library with Ligation Sequencing Kit (SQK-LSK109; Oxford Nanopore Technologies) according to the manufacturer’s instructions. The library was sequenced with PromethION (Oxford Nanopore Technologies) using R9.4.1 flow cells (Oxford Nanopore Technologies). The same DNA was also sequenced using a NovaSeq (250 bp, paired-end) (Illumina).
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