iPSCs were cultured and dissociated as described for nEBs above. nEBs were formed in hES media without FGF for 16 days with media changes every two days. nEBs were harvested and RNA was collected using a MicroRNAeasy kit (Qiagen, 217004). Lineage scorecard was calculated for nondirected EB differentiation using the NanoString probe set described by Bock et al. after quantification using single-molecule imaging on an nCounter System according to the manufacturer’s instructions by the Meissner Lab at Harvard University.37
