To dissociate hCS and hSS for single cell profiling, we adapted a previously published protocol used for primary human fetal brain tissue44. Briefly, up to 6 spheroids were chopped using a #10 blade and then incubated in papain enzyme solution (27.3 U/ml; Worthington), EBSS (1×, Sigma), 0.46% Sucrose (Sigma), 26 mM NaHCO3 (Sigma), 0.5 mM EDTA (Sigma) at 37°C for 70 min in an incubator (5% CO2). The digested spheroids were then washed and carefully triturated in a trypsin inhibitor solution EBSS, 0.46% Sucrose (Sigma), 26 mM NaHCO3 (Sigma), 15–30 mg Trypsin Inhibitor (Sigma). After centrifugation, the pellet was resuspended in 0.2% BSA diluted in PBS and supplemented with Y-27632 (10 μM; EMD Chemicals) and the cells were used for FACS.
