Gaspak ez anaerobe pouches

For the TPE spot assay, strains integrated with the URA3 gene at TELV07L were used (see Table S3; kindly provided by Paul Kaufman). Gene knockouts were created using insertion of targeted PCR cassettes amplified from the pFA6a vector series (Longtine et al, 1998 (link)). Cells were grown overnight in YPD medium at 30°C and 0.1 OD units of the cultures were serially diluted and spotted on YPD plates (control) and 5-fluoroorotic acid (5-FOA) plates. The plates were observed and imaged for 2 d to analyze the growth pattern. For growth analysis of single and double mutant strains under aerobic and hypoxic conditions, yeast strains were grown overnight in YPD, diluted to OD₆₀₀ ∼ 0.2 the next day, and grown to log phase. 0.1 OD₆₀₀ units of the culture were serially diluted and spotted on YPD plates. For hypoxic conditions, the plates were incubated at 30°C in BD GasPak EZ anaerobe pouches. The plates were observed and imaged for 2 d for aerobic conditions and 8 d for hypoxic conditions. The data were quantified as described (Petropavlovskiy et al, 2020 (link)). Briefly, the mean gray value quantitation was performed using ImageJ. The background for each image was adjusted using the sliding paraboloid function. The mean gray value was calculated for one dilution across multiple strains and background subtraction was performed.