An ENU mutagenesis/whole exome deep sequencing pipeline enables rapid identification of causative mutations in mice with defects in epigenetic gene silencing. A schematic overview of the ENU mutagenesis gene discovery pipeline is presented. The major components of the screen are described in the figure. Briefly, male FVB/NJ mice carrying an epigenetically sensitive GFP transgene (Line3) were treated with ENU and mated with female Line3 mice. Offspring were screened for a shift in the percentage of GFP expressing erythrocytes using flow cytometry. Putative mutants were mated with Line3 mice for four generations to test for heritability and reproducibility of the GFP expression, and to reduce the number of non-causative ENU mutations within the genomes. Linkage analysis was carried out on the offspring from two generations of backcrosses between putative mutants and C57BL/6J mice, which also carry the GFP transgene (Line3C). Causative mutations were identified by whole exome deep sequencing, or gene candidate sequencing on individuals that had been maintained on the Line3 background for at least seven generations. FACS, fluorescence-activated cell sorting.