Primesurface 96 well plates

mESCs (EB5; passages 35–45) and CTIP2:GFP KI mESCs (passages 11–21) were maintained on a mitotically inactivated mouse embryonic fibroblast feeder layer in KnockOut DMEM (Thermo Fisher Scientific) supplemented with 20% (vol/vol) Fetal Bovine Serum (FBS; Merck), 1% PS, 0.1 mM 2-ME, 2 mM L-Gln, 2,000 U ml⁻¹ Leukocyte Inhibitory Factor (LIF; Merck) and 1% (vol/vol) Nucleosides (Merck). The culture medium was replaced with fresh medium every day. To induce cortical neurons, the mESCs were replated in prime surface 96-well plates (Sumitomo Bakelite) at a density of 9,000 cells per well in differentiation medium containing GMEM (Thermo Fisher Scientific) supplemented with 10% (vol/vol) KnockOut Serum Replacement (KSR; Thermo Fisher Scientific), 0.1 mM MEM non-essential amino acids solution (NEAA; Thermo Fisher Scientific), 0.1 mM 2-ME, 1 mM sodium Pyruvate solution (Pyruvate; Merck) and 2 mM L-Gln. 10 μM SB-431542 (Merck), which is a TGF-β receptor inhibitor, and 20 nM WNT-C59 (Collagen Technology, San Diego, CA, USA), which is a WNT inhibitor, were added by day 6. On day 7, we switched the medium from GMEM to DMEM/F12 (Fujifilm) supplemented with 0.1 mM 2-ME, 2 mM L-Gln, 1% N2 supplement and 2% B-27 supplement. Half of the medium was replaced with fresh medium every 3 days.