GSM9326596: M.CviPI wild-type replicate 2, 120 min; Saccharomyces cerevisiae; OTHER
Cell pellets were permeabilized and flushed with two washes of 500 µL of 5× TE (50 mM Tris-HCl pH 8.0, 5 mM EDTA) + 2% SDS. Next, to remove the SDS, they were washed twice more with 500 µL 5× TE, then resuspended in 450 µL 5× TE + 15 mM 2-mercaptoethanol. To digest cell walls, 50 µL lyticase (Sigma-Aldrich L2524, 25,000 units/mL) was added, mixed gently by inversion, then incubated at 37°C for 10 min. To stop the reaction, 50 µL 20% SDS was added, mixed gently, and incubated at room temperature for 5 min. DNA was isolated by the addition of 110 µL 5 M potassium acetate, followed by two 1× volume chloroform extractions, precipitating and pelleting with 0.7× volume isopropanol, and washing with 70% ethanol. The resulting pellet was air-dried and dissolved in 100 µL 10 mM Tris-HCl pH 8.0, 0.1 mM Na-EDTA, 0.4 mg/mL RNase A and incubated at 37°C for 2 h. Further purification of DNA was performed with the PureLink Genomic DNA Mini Kit (Invitrogen K182002). 10 µL Proteinase K and 10 µL RNase A, supplied with the kit, were added to each sample and incubated at room temperature for 2 min. 100 µL PureLink Genomic Lysis/Binding Buffer was added to each sample, mixed gently by inversion, and incubated at 55°C for 10 min. Finally, 100 µL 100% ethanol was added to each sample and mixed gently by inversion before proceeding to the “Binding DNA” step of the manufacturer-provided protocol and completing the extraction as per instructions. Genomic DNA samples were barcoded with the Native Barcoding Kit (SQK-NBD114.24) from Oxford Nanopore Technologies (ONT) as per manufacturer instructions and sequenced with R10.4.1 flow cells on a MinION Mk1C instrument running MinKNOW 24.11.8 (ONT).