RNA was extracted from undifferentiated pluripotent stem cell cultures (2 BL6 replicates, 2 CAST replicates) following feeder depletion and from pluripotent stem cell-derived motor neurons (4 BL6 replicates, 1 CAST replicate) following 7 days of culture on laminin. For pluripotent stem cell samples, we used the RNAqueous™-Micro Total RNA Isolation Kit (Thermo Fisher Scientific). For neuron samples, we used the RNAeasy-Micro Total RNA Isolation Kit (Thermo Fisher Scientific). In each case we used on-column DNase treatment (QIAGEN, Hilden, Germany). RNA samples were processed into mRNA libraries and sequenced on an Illumina NovaSeq 6000 Sequencing System, yielding ~20M paired-end 150 bp reads per sample. Read-mapping was as above. Normalized read counts from these stem cell and neuronal RNA-seq datasets are reported in Table S2 .
For principal component analysis, we filtered expression data from purebred stem cell and neuron samples (Skelly et al. [2020 (link)] and data generated in this study;Table S2 ) to remove non-coding RNAs and mitochondrially-encoded genes. For the remaining genes, we calculated log2(TPM+1) values for input into the prcomp module in R.
For principal component analysis, we filtered expression data from purebred stem cell and neuron samples (Skelly et al. [2020 (link)] and data generated in this study;
