Lentiviral production and fibroblast infections were performed as described previously13. Briefly, primary human fetal or postnatal fibroblasts were plated and infected with concentrated lentiviral particles and polybrene (8 µg/µL) in fresh MEF media. Viral media was removed after 16–24 hours and replaced with MEF media containing doxycycline (dox, 2 µg/µL). After 24–48 hours, media was changed to N3 media containing dox (2µg/µL). For human ES cell infections, H9 human embryonic stem cells were switched into N3 media containing polybrene (2 µg/µL) 24 hours after re-plating, and concentrated lentiviral particles were added. After 16–24 hours, cultures were switched into N3 media containing dox (2 µg/µL) and changed daily before dissociation. Forty-eight hours after the initial addition of dox, Ara-C (4 µg/µL) was added to the media to inhibit proliferation of uninfected ES cells until analysis 6 days after the addition of dox. All chemicals were purchased from Sigma-Aldrich™ if not otherwise specified.
