Illumina sequencing of Pyricularia oryzae isolate KEN54-20 M73
Mycelia from one-week-old plates of P. oryzae isolates KEN54-20 and avr-Rmo1 mutants were collected, ground with liquid nitrogen to a very fine powder and transferred into 1.5 mL microcentrifuge tube until about two thirds full. 500 uL of CTAB buffer (0.2 M Tris-HCl pH 7.5, 50 mM EDTA, 2 M NaCl, 2% CTAB) pH 7.5 were added and samples were incubated at 65C for 30 min, shaking every 10 min. Subsequently, 500 uL of chloroform:isoamyl alcohol (24:1) were added and samples were incubated for 30 min under constant shaking at 300 rpm, followed by centrifugation at 16000xg for 10 min. The aqueous phase (supernatant) was transferred into a new 1.5 mL microcentrifuge tube and 500 uL of chloroform:isoamyl alcohol (24:1) were added, mixed for five min, and then centrifuged at 16000xg for 10 min. The top aqueous phase was transferred to a new tube and 1mL of ice-cold isopropanol was added and mixed. Samples were incubated at -20C for two h and then centrifuged at 16000xg for 10 min. The supernatants were discarded. DNA pellets were allowed to drain for five min and then completely resuspended in 500uL of sterile water. 50 uL of 3 M NaOAc were added with 1 mL of ice-cold 100% ethanol, followed by incubation at -20C for one hour and centrifugation at 16000xg for 20 min. The supernatants were discarded and 400 uL of ice-cold 70% ethanol were added. The samples were centrifuged at 16000xg for 5 min, the supernatants were discarded and the pellets were allowed to air-dry. DNA samples were resuspended in 100 uL of TE+RNAse A (Thermo Fisher) and stored at 4C. Concentration of DNA samples were measured using the Qubit dsDNA HS Assay Kit (Thermo Fisher). DNA samples were submitted for library preparation and whole genome sequencing by Illumina to Novogene.