Endogenous Tagging of NG2 Protein in HEK-293A Cells

We generated HEK-293A cells endogenously tagged with the split NG2 protein at the Pfn1 and SHIP2 (INPPL1) loci, following a previously published general protocol (57 (link)). Briefly, we electroporated a Platinum Cas9 (Thermo Fisher Scientific#B25640)–gRNA complex with a single-stranded HDRT (IDT) into our stably expressing HEK293^NG2-1-10 cells. The HDRT is composed of 70 bp homology arms to align with the gene of interest, the NG2 11th β-strand insertion sequence, and a flexible linker. HDRT and gRNA sequences for INPPL1 are provided in Table S2. HDRT and gRNA sequences for Pfn1 are from OpenCell database. Following electroporation, we screened cells for fluorescence using our confocal microscope and subsequently used fluorescence-activated cell sorting (University of Pittsburgh Flow Cytometry Core) to isolate and expand NG2-positive cells.