Extended Data Fig. 4. In situ dose-dependent transduction profiling of barcoded AAV pools with USeqFISH.
a, Schematic of experimental design. A pool composed of AAV-PHP.eB carrying the viral genome with five unique barcodes was delivered at different doses (1011, 1010, 109, 108 and 107 vg for each barcode per animal). After delivering the pool to mice via retro-orbital injection, USeqFISH was applied to the harvested brain tissue to detect the barcodes of each variant in parallel. b, Immunohistochemistry (IHC) with GFP antibodies, labeling spGFP(1–10) used as the coding sequence of the barcoded viral genome, showed broad expression of the injected pool across brain regions, including the cortex (red outlined box), thalamus (green outlined box), and cerebellum (blue outlined box). c, Representative images of the expression of the 5-dose pool. Viral transcript spots are indicated with yellow arrows in the images of lower doses (109, 108, and 107 vg). d, The transduction rate of AAV-PHP.eB at each dose acquired from five mice (indicated by different colors; the mean of each dose is indicated by the black line; mean ± s.e.m.). e, The cumulative distribution of viral RNA spot number in each transduced cell at each dose (Kolmogorov-Smirnov test).