For ONT sequencing, we extracted genomic DNA from muscle using the phenol-chloroform-isoamyl alcohol extraction, followed by isopropanol precipitation. The DNA was further purified with NucleoBond AXG 100 columns with the NucleoBond Buffer Set IV (Macherey-Nagel, Germany). ONT libraries were prepared with the Ligation Sequencing Kit (SQK-LSK109; Oxford Nanopore Technologies, UK) and were sequenced using multiple R9.4.1 flow cells on an ONT GridION platform. The raw .fast5 files were base-called by Guppy v4.0.11 with the high-accuracy mode and quality filtering. Reads that were shorter than 1 kb were discarded using SeqKit v0.12.0 (Shen et al. 2016 (link)). The length-filtered ONT long reads were used in the downstream analyses. Supplementary Table S3 summarizes the filtered genomic reads generated in this study.
Nucleobond buffer set 4
Manufactured by Macherey-Nagel
Sourced in Germany
NucleoBond Buffer Set IV is a collection of buffers used in the purification of plasmid DNA. The set includes a range of solutions designed to facilitate the efficient extraction and isolation of plasmid DNA from bacterial cultures.
Automatically generated - may contain errors
2 protocols using nucleobond buffer set 4
1
ONT Sequencing of Genomic DNA from Muscle
2
Genomic DNA Isolation and Sequencing of KOR1 Cells
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About 8 mL wet volume of KOR1 cells were harvested by centrifugation at 5000×g for 1 min. The cell pellet was washed once with distilled water, frozen in liquid nitrogen, and then milled using a mortar. Genomic DNA was purified from the frozen cell powder using NucleoBond Buffer Set IV and NucleoBond AXG 100 Column (MACHEREY-NAGEL, North Rhine-Westphalia, Germany) according to the manufacturer’s manual. Library preparation and whole genome sequencing were performed by Takara Bio (Shiga, Japan). The sequence data of KOR1 were analyzed using CLC Genomics Workbench 12.0 (CLC bio, Aarhus, Denmark), and compared to the genomic sequence of JSC4 determined in the previous study22 (link).
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