Log phase cultures of cells were harvested and labeled with NHC-Biotin as previously described [16]. Labeled populations were transferred to YEPD and incubated for 30°C, 2 hours to ensure that most labeled cells were mothers (have completed at least one cell division). Cells were counted and used to inoculate 1.5 liter cultures of YEPD +1 µM estradiol +100 µg/ml ampicillin at a density of 2×104 cells/ml. Cultures were incubated at 30°C, shaking at 100 rpm for the indicated times before purification. Cells were harvested by centrifugation, resuspended at 6×108 cells/ml in RNAlater (Ambion) and fixed at room temperature, 45 minutes. Cells were harvested by centrifugation and resuspended at 2×108 cells/ml in PBS +2 mM EDTA and incubated with 1/20 volume streptavidin beads (Miltenyi Biotec), 4°C, 30 minutes. Cells were harvested by centrifugation and resuspended in 4 ml 40 mM Tris HCl pH 7.4 and layered onto Percoll Plus gradients (GE Healthcare). Gradients were spun at 4°C, 20 minutes at 2000 RPM in a GS-6R tabletop centrifuge (Beckman). A brown, flocculent layer of cell debris was removed from the top of
