ES cells containing a Nanog-Luciferase construct31 were infected in biological duplicate and monitored after 7 days. Luciferase activity was measured using Bright-Glo (Promega). All reagents and cells were equilibrated to room temperature. 100ul Bright-Glo solution was added to each plate well. Plates were incubated in the dark at room temperature for 10 minutes and luciferase was measured on a plate reader. The luciferase units were normalized to the control hairpins and a z-score compared to the negative controls (excluding luciferase hairpins) was computed. For each hairpin, we computed a Z-Score relative to the negative control hairpins and identified hits reducing Luciferase levels more than 6 standard deviations (Z<−6) for both independent replicates. In all cases we were able to identify a significant reduction in luciferase levels when using distinct hairpins targeting luciferase. To exclude hits that were due to an overall reduction in proliferation (which would also cause a reduction of Nanog positive cells in this read-out) we excluded all hairpins that caused a reduction in proliferation as measured by AlamarBlue incorporation (described below). AlamarBlue incorporation was measured in the same cells immediately before reading out Nanog-Luciferase levels.
