Nucleobond axg 100 column

The genomic DNA from four vaa-mutants, two oppA-mutants and from the M. hominis PG21 parental strain was extracted using NucleoBond® AXG100 columns (Macherey-Nagel, Düren, Germany) and the NucleoBond® buffer Set III (Macherey-Nagel). The genomes were sequenced using paired-end V2 2X250 bp sequencing on MiSeq Illumina apparatus (San Diego, CA, USA) after generating the genomic DNA libraries using the of Nextera XT DNA Library Preparation Kit (Illumina). About 160,000 to 200,000 paired reads were obtained for each genome. Data processing including quality check, trimming, alignment with BWA (Galaxy Version 1.2.3) and variant calling using Varscan (Galaxy Version 0.1) was performed using Galaxy at https://usegalaxy.org/ [20 ].

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 study^{22 (link)}.

Fresh leaves of Koshu were collected at the Yamanashi Fruit Tree Research Station (Yamanashi, Japan). Approximately 90 g of fresh leaves was ground in liquid nitrogen, and the fine powder was transferred to a suspension buffer (10 mM Tris-HCl at pH 9.5, 80 mM KCl, 10 mM ethylenediaminetetraacetic acid, 10 mM spermidine, 0.5 M sucrose, 0.5% Triton X-100, and 0.15% 2-mercaptoethanol). After the powder was filtered through Miracloth (Merck Millipore), the filtrate was centrifuged at 2,000 × g for 10 min. The collected pellet was resuspended in the suspension buffer, and genomic DNA was extracted using the CTAB method (Doyle and Doyle, 1987 ). The extracted DNA was purified using a NucleoBond AXG 100 column (Macherey-Nagel, Düren, Germany). The purity and concentration of the extracted DNA were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, United States) and a Qubit fluorometer (Thermo Fisher Scientific) with a Quant-iT dsDNA BR Assay Kit (Thermo Fisher Scientific). Genomic DNA fragments were visualized by agarose gel electrophoresis and pulsed-field gel electrophoresis.