URA3 reporter gene assay for silencing.

Cells were grown to saturation overnight in 2 ml of YPD medium containing hygromycin B (to select for plasmids). The cells were then pinned onto plates with three different media: complete supplement mixture (CSM) containing hygromycin B (to assay overall growth), CSM containing hygromycin B and lacking uracil (to select for cells expressing URA3), and CSM containing uracil and 5-fluoroorotic acid (5FOA) to select for cells not expressing URA3 (20). The cells were pinned in a 5-fold dilution series, and the plates were imaged on day 3 of growth.