iPSCs were grown on MEFs or Matrigel to confluence. iPSCs were dissociated with Accutase (Invitrogen, A11105-01). 3,000–6,000 single cells were placed in Aggrewell 400 plates (Stemcell Technologies, 27945) and incubated in hES media without FGF for 24 hours. Aggregates were released and passed through the 40 µm cell strainer to remove single cells. 1,000 aggregates/well were plated in an ultra-low adherence 6-well plate (Corning, 3471) in Neural Induction medium on day 0. Neuralized embryoid bodies (nEBs) were fed every other day and harvested on days one and eleven for further analysis. RNA was isolated from EBs using the MicroRNAeasy kit (Qiagen, 217004).
