Supplementary Figure 6: Generation of the CUX2-CreER^T2 conditional reporter hPSC line and early generation of CUX2+ neurons in both P1S5D and P8S10D treated cells.

(a) Design of the homology donor targeting the CUX2 first exon. The selection cassette was excised upon expression of Flp recombinase after transgenesis. The location of primer sequences to confirm targeting is shown. (b) Schematic illustration of the targeted alleles. The transgenic lines express CreER^T2 from the CUX2 locus and the FLEX-tdTomato conditional reporter under the CAG promoter at the AAVS1 safe harbor locus. Expression of CUX2 in the presence of 4OHT induces recombination at the reporter locus and tdTomato expression. (c) PCR confirmation of targeted transgenesis. Lanes 1-2 confirm 5’ and 3’ CreER^T2 insertions at the CUX2 genomic locus, respectively, while lanes 3-4 confirm 5’ and 3’ CAG-FLEX/tdTomato insertions at the AAVS1 genomic locus, respectively. (d) Genomic DNA sequencing at the CUX2 locus confirms successful targeting of one allele and a wild-type non-targeted allele. (e) tdTomato positive post-mitotic neurons at day 70 in culture after 4OHT induction (i). Typical pyramidal morphology and lengthy projections can be observed upon higher magnification (ii). (f) CUX2+ neurons with mature morphologies observed in P1S5D and P8S10D culture at day 33 of differentiation. (g) Pyramidal morphology at day 33 suggests cortical projection neuron identify of P1S5D and P8S10D neurons. Scale bars in e (i) represents 100 μm, while those in others represent 50 μm.