Expression of synaptic function genes in iPSC-derived induced human neurons
To determine expression levels of neuron-associated genes in induced human neurons, cultures of Ngn2-induced neurons grown on mouse glia were dissociated and assayed for nuclear RNA using the 10X Multiome kits.  Associated ATAC data were not evaluated in this study.  A mixture of 10 iPSC cell lines, 6 from diagnosed participants with alcohol use disorder (AUD) and 4 unaffected controls, were infected with lentiviral vectors expressing doxycycline-inducible Ngn2.  Following drug selection for successful transduction, cells were plated onto primary mouse glial cells and allowed to mature for approximately 4 weeks.  Cells were harvested with Accutase and Papain, then processed using standard 10X Genomics protocols using the Multiome kit.  After importing into Seurat and clustering, excitatory neuron cells were selected and deconvolved to identify the source participant by matching SNPs from earlier GWAS studies.  Tables of genes x barcode were then aggregated into genes x cell line matrices, which were loaded into DESeq2 for scaling and normalization.  For this study, only processed data from 3 unaffected control subjects are reported.  Results demonstrate robust expression of a series of synaptic receptor, channel, and associated signaling genes. Overall design: The goal was to evaluate expression levels of selected synaptic genes to confirm that the cultured induced neurons were likely to respond to stimuli and fire action potentials, as shown in our study.