Esc grade fetal bovine serum

mESC lines were cultured in 2-inhibitors-based medium [advanced Dulbecco's modified eagle medium (DMEM)/F12:Neurobasal (1:1) medium (Gibco), supplemented with 2.5% ESC-grade fetal bovine serum (v/v, Corning), N2 (Gibco), B27 (Gibco), 2 mM L-glutamine (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1000 U/ml leukemia inhibitory factor (Millipore), 3 μM CHIR (BioVision) and 1 μM PD0325901 (Sigma-Aldrich)] on 0.1% gelatin-coated (Millipore) plates at 37°C and 8% CO₂. In vitro differentiation of mESCs to MNs has been described previously (Tan et al., 2016 (link); Wichterle et al., 2002 (link); Wichterle and Peljto, 2008 (link)). Briefly, embryoid bodies (EBs) were obtained by plating trypsinized (Gibco) mESCs in AK medium [advanced DMEM/F12:Neurobasal (1:1) medium (Gibco), 7% KnockOut SR (v/v) (Gibco), 2 mM L-glutamine, 0.1 mM β-mercaptoethanol and penicillin–streptomycin (Gibco)] at 37°C, and 5% CO₂ (day −2). On day 0, EBs were split 1:2 and AK medium was replenished and supplemented with 1 μM all-trans RA and 0.5 μM smoothened agonist (SAG) (Millipore, 566660). TF induction was performed by adding 3 μg/ml of Dox (Sigma-Aldrich, D9891) on day 2. For RNA-seq and ATAC-seq experiments, 3.5×10⁵ mESCs were plated in 100 mm suspension dishes (Corning). For ChIP-seq experiments, 3-3.5×10⁶ mESCs were plated in 245 mm×245 mm square dishes (Corning).