Tetrahymena Mating Induction Protocol

Wild-type Tetrahymena strains CU428.2 (mating type VII, RRID:TSC_SD00178), B2086 (mating type II, RRID:TSC_SD01627), and SB210 (mating type VI, RRID:TSC_SD00703) were obtained from the Tetrahymena Stock Center, Cornell University (https://tetrahymena.vet.cornell.edu/). ΔSPO11 (Mochizuki et al., 2008 (link)), SPO11i (Howard-Till et al., 2013 (link)), and Asf1-GFP (Garg et al., 2013 (link)) strains were constructed previously. Cells were grown at 30°C in SPP medium containing 1% proteose peptone (Becton Dickinson, Sparks, MD, USA), 0.1% yeast extract (Becton Dickinson), 0.2% glucose (Sigma-Aldrich, St. Louis, MO), and 0.003% EDTA-Fe (Sigma-Aldrich). To make them competent for mating, cells at mid-log phase (approximately 10⁶/ml) were washed with 10 mM Tris-HCl (pH 7.4), resuspended in 10 mM Tris-HCl (pH 7.4), and starved at 30°C for ~18 hr to starve. To induce mating, equal numbers of cells of two different mating types were mixed together and incubated at 30°C.