Extended Data Fig. 10: Strategy for siRNA-induced silencing at the ura4∆::ade6⁺ locus.

a, The ura4⁺ coding sequence was replaced with ade6⁺ to generate a ura4Δ::ade6⁺ allele in cen::ade6⁺ leo1Δ ade6-M210 cells. Formation of red colonies on low adenine medium indicated ura4Δ::ade6⁺ silencing. b, cen::ade6⁺ leo1Δ ura4Δ::ade6⁺ OFF cells were crossed to cen⁺ ura4Δ::ade6⁺ ON cells to demonstrate that the resulting ura4Δ::ade6⁺ OFF state is stable in the absence of the cen::ade6⁺ siRNA trigger or the leo1Δ enabling mutation. c, d, Crosses showing that the ura4Δ::ade6⁺ OFF epigenetic state depends on Ago1 (c) and Clr4 (d). e, Cross for generating an epe1Δ ura4Δ::ade6⁺ OFF epiallele (top) and comparison of RNAi-independent (TetR–Clr4-I-induced) and RNAi-dependent (cen::ade6⁺-induced) ade6⁺ OFF epialleles. The same results were obtained with independent clones.