Diploid cells were grown to saturation overnight in YC media lacking adenine and methionine. Cells were used to inoculate YEPD cultures, which were grown to log phase by incubation with shaking, 30°C for 3 hours. Cells were counted and used to inoculate 25 ml YEPD +1 µM estradiol cultures at 2×104 cells/ml and incubated with shaking, 30°C for 95 hours. At indicated times, samples were harvested, washed, and plated to lead nitrate media. Sample volumes were adjusted appropriately to maintain a colony density of ∼500–1000 colonies/150 mm plate. Plates were incubated at 30°C for 3 days and colonies were counted using a Geldoc XR+ imaging system (Biorad). Plates were further incubated at room temperature for 2–3 days for color development before scoring for half sectors. Reciprocal and non-reciprocal colonies were counted separately, and rates of total half sectors were calculated as (2*reciprocal + non-reciprocal/total colonies) [17].
