Workflow for Assessing Transcriptional Regulation

Transfections and viral infections were performed as previously described^{40 (link)}. For acute analysis of gene expression changes, RNA was isolated from GFP^high cells FACS-sorted 48 hours after transfection with MigR1-N1ICD or Rest-IRES-GFP or the empty vector control. Viral transductions of N1ICD or Rest were used to generate adherent non-NE cells from NE cells, a process which takes about 1–2 weeks. The cells were then expanded and collected for immunoblot analyses. For isolation of Rest-knockout clones, sgRNA-infected cells were selected with puromycin (2 μg/ml) for 4 days and single cells were sorted into individual wells in 96-well plates by FACS. After two weeks, clones were picked and clones with biallelic frameshift mutations resulting in premature truncation of the translated protein were verified by TOPO® PCR cloning (Thermo Fisher Scientific) and Sanger sequencing.