Extended Data Figure 2. Brain and Cortical Organoid Generation Protocols.

a) Cortical organoid protocols utilizing different levels of directed differentiation were evaluated using scRNA-seq and immunohistochemistry. Stem cells were expanded on matrigel, dissociated to single cells, and re-aggregated in v-bottom low adhesion plates. Small molecules were used to promote forebrain induction and after 18 days moved to 6 well plates onto an orbital shaker. Organoids were maintained in culture and collected from weeks 3-24. b) Protocol schematics for other methods used to differentiate whole brain and cortical organoids, which have published single cell data. Publicly available data was utilized for comparative analyses with our collected data.