RNA-seq of AMFR mutant human in vitro differentiated neural stem cells

We report transcriptome analysis of human in vitro differentiated neural stem cells (derived from H9 human embryonic stem cells), comparing wild type and AMFR homozygous knock-out mutant cells. To model a novel genetic disorder caused by bi-allelic loss-of-function variants in AMFR and to study the effects on the global transcriptome caused by the absence of AMFR, we generated knock-out mutant cells, by CRISPR-Cas9 genome editing, targetting exon 4. This resulted in frame shift mutations with absence of full length AMFR confirmed by Western blotting. Knockout mutant and wild type cells were then compared to determine differentially expressed genes. For the RNA-seq experiments, two independent wild type cultures, three independent clones harboring the homozygous AMFR mutation were used. All cell lines were cultured in two technical replicates, and sequenced at passage 5 of NSC differentiation.