Experimental design. Standard mutagenesis protocols were modified by (1) selecting for unc-22/+ animals (twitching in 1% nicotine) in the F1 generation to ensure that the gametes had been effectively exposed to the mutagen; (2) counter-selecting in the F2 generation to generate an unc-22(+) background for the mutant strains; and (3) clonally propagating independent lines for 10 generations, driving strains to homozygosity to simplify variant calling and to create uniform, stable strains for further manipulations.