V6.5 ESCs or Nanog-Luciferase ESCs were plated at a density of 5000 cells/well (8-day time point) or 25,000 cells/well (4-day time point) in 100ul mES media onto pre-gelatinized 96-well dishes (VWR; BD356689). Cells were infected with 5ul of a lentiviral shRNA stock and incubated at 37°C for 30 minutes. Puromycin resistant DR4 MEFs (GlobalStem; GSC-6004G) were then added to the plates at a density of ~6000 cells/well and incubated overnight at 37°C, 5% CO2. After 24 hours, all media was removed from the cells and replaced with media containing 1ug/mL puromycin. Media was then changed every other day with fresh media containing 1ug/mL puromycin. The end-point depended on the assay and was either 4-days post infection (knockdown validation and microarrays) or 8-days (reporters and qPCR of marker genes).
