The Tet-inducible-Cdx2-Flag ES cell line (iCdx2 cells) was obtained from NIA mouse ES cell bank27 (link). iCdx2 cells were maintained on feeder cells in DMEM supplemented with 15% fetal bovine serum (Gibco, Thermo Fisher Scientific), 1% non-essential amino acids, 2 mM l-glutamine, 1,000 units of mLIF (EMD Millipore), 0.1 mM β-mercaptoethanol (Sigma), antibiotics and 2 μg/ml doxycycline (Sigma). When starting differentiation44 (link), iCdx2 cells were selectively seeded on gelatin-coated plates after stepwise elimination of the feeder MEF, then Cdx2 overexpression was induced by removing doxycycline. The cells were split at day 1 after doxycycline withdrawal and cell samples were collected in the following days.
Mouse ES cells in 2i/L were cultured in commercially available 2i medium kit (Milipore, SF016-200). a2i/L medium contains a 1:1 mixture of DMEM/F12 supplemented with N2 (Invitrogen) and Neurobasal media with glutamine (Invitrogen) supplemented with B27 (Invitrogen), 1X Pen/Strep (Invitrogen), 1000 units of LIF (Milipore), 1.5 μM CGP77675 (Tocris) and 3 μM CHIR99021 (Tocris). PKCi/L medium contains a 1:1 mixture of DMEM/F12 supplemented with N2 (Invitrogen) and Neurobasal media with glutamine (Invitrogen) supplemented with B27 (Invitrogen), PenStrep (Invitrogen), 1000 unites of mLIF (Milipore) and 5 μM Gö6976 (Tocris).
Mouse ES cells in 2i/L were cultured in commercially available 2i medium kit (Milipore, SF016-200). a2i/L medium contains a 1:1 mixture of DMEM/F12 supplemented with N2 (Invitrogen) and Neurobasal media with glutamine (Invitrogen) supplemented with B27 (Invitrogen), 1X Pen/Strep (Invitrogen), 1000 units of LIF (Milipore), 1.5 μM CGP77675 (Tocris) and 3 μM CHIR99021 (Tocris). PKCi/L medium contains a 1:1 mixture of DMEM/F12 supplemented with N2 (Invitrogen) and Neurobasal media with glutamine (Invitrogen) supplemented with B27 (Invitrogen), PenStrep (Invitrogen), 1000 unites of mLIF (Milipore) and 5 μM Gö6976 (Tocris).