The NIL (Ngn2-Isl1-Lhx3)-V5 inducible ESC line was previously described (Mazzoni et al., 2013 (link)). All the inducible ESC lines were grown in 2-inhibitors medium (Advanced DMEM/F12:Neurobasal (1:1) Medium (Gibco), supplemented with 2.5% ESC-grade fetal bovine serum (vol/vol, Corning), N2 (Gibco), B27 (Gibco), 2mM L-glutamine (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1000 U/ml leukemia inhibitory factor (Millipore), 3μM CHIR (BioVision) and 1 μM PD0325901 (Sigma). To obtain Embryoid bodies (EBs) ESC were trypsinized (Gibco) and 3 × 105 cells were plated in each 100 mm dish in AK medium (Advanced DMEM/F12:Neurobasal (1:1) Medium, 10% Knockout SR (vol/vol) (Gibco), Pen/Strep (Gibco), 2mM L-glutamine and 0.1 mM 2-mercaptoethanol) (day −2). After 48 hr, EBs were passed 1:2, and the inducible cassette was induced by adding 3 μg/ml of Doxycycline (Sigma) and/or 1 μM all-trans retinoic acid and/or 0.5 μM smoothened agonist (SAG) (Millipore, 566660). Differentiating EBs were washed three times with PBS, dissociated with Trypsin, and pipetted into single-cell suspensions. After 48 hr, cells were preserved in methanol (Alles et al., 2017 (link)) before processing for single-cell profiling (below).
Kong W., Fu Y.C., Holloway E.M., Garipler G., Yang X., Mazzoni E.O, & Morris S.A. (2022). Capybara: A computational tool to measure cell identity and fate transitions. Cell stem cell, 29(4), 635-649.e11.
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