Monolayer and embryoid body neural induction protocol

Cells carrying inducible shRNAs against SMARCB1 were cultured for 3 days in the presence or absence of 1 µg/ml doxycycline. Cells were then collected in Gentle Cell Dissociation Reagent for 10 min at 37°C and dissociated into single cells. The remainder of the protocol was performed as per the STEMCELL Technology instructions for neural induction using STEMDiff Neural Induction Media (#05835), with minor modifications. Specifically, cells were plated at a density of 1.5 × 10⁵ cells/cm², as this density provided the highest efficiency of induction. At 6 days, cells were collected for RNAseq, ATACseq, or processed for immunohistochemistry. The monolayer protocol was performed three times with three biological replicates each, with similar results for each performance.

EBs were formed from control and SMARCB1 KD cells (following 3 days of dox treatment). The EBs were 1,000 cells each and were generated in AggreWell400 plates (StemCell Technologies #34421). After 24 hr, the EBs were transferred to ultra-low-adherence dishes (Corning #3471), following the StemCell Technologies protocol. The EBs were cultured in AggreWell EB Formation Medium (StemCell Technologies, #05893), with media replaced daily by allowing the EBs to settle in 15 ml conical tubes for 10 min prior to media aspiration. On Day four following EB formation, 1 µM all-trans retinoic acid was added to the media, whereupon the EBs were cultured for an additional 4 days prior to collection for isolation of RNA. The EB experiment was performed 5 times with 2–3 replicates per control/SMARCB1 KD condition for each experiment. Similar morphological effects of SMARCB1 KD were observed for each performance, and qPCR analysis was performed on replicates from one performance.

For both monolayer and EB experiments, RNA was isolated using Qiagen RNAeasy kits (#74104), and cDNA was generated using an iScript cDNA Synthesis Kit (BioRad, #1708891). The primers used for the analysis of neural and pluripotency markers are available in Supplementary file 6.