In vitro human H9 ESC-derived microglia scRNA seq
Given that the ?-secretase substrate proteome identified in H9MG reveals its central role in microglial signaling-regulatory events, we reasoned that investigating the overall profile of the transcriptomes of the microglia would provide a good integrated read-out for the overall context-dependent effect of blocking the processing of >85 ?-secretase substrates. Although the alteration in tonic signaling caused by ?-secretase inhibition does not change the overall cell state, i.e. the cells with ?-secretase deficiency do not cluster dramatically differently in the UMAP plots (Figure 4E-F and J-K), the tonic signaling is robust as it remains discernible in the background of a strong LPS-induced response (Figure 4H and M). Overall design: We analyzed the single-cell transcriptome of in vitro microglia in the presence and absence of the ?-secretase inhibitor Semagacestat to see how the inhibition of the processing of these proteins affect gene expression.  We performed a second experiment in parallel asking whether a strong ?-secretase independent signal could overcome the predicted tonic signaling. To this end, we treated cell cultures for 6 hours with lipopolysaccharide (LPS). Therefore, a total 12 samples in 4 experimental conditions (Ctrl, LPS, Sema, SemaLPS, with 3 biological replicates in each condition) were labeled with unique Hashtag antibodies and equally pooled. The pooled cells were then equally devided into 2 libraries (GEO samples) for scRNA seq.