AAV-Zombie on cultured cells

Detection of AAV genomes in situ can facilitate understanding of AAV transduction and processing. This protocol enables detection of AAV genomes and concatemers in cultured cells and is based on the Zombie method published in Askary et al., Nat Biotechnol 38: 66. AAVs carrying barcoded genomes are delivered to cells. Following fixation, a phage RNA polymerase is used to transcribe the barcode in situ, yielding barcoded transcripts that can be detected with RNA FISH. This protocol is compatible with either ssAAV or scAAV genomes. In the former case, detected genomes will be primarily nuclear and detectable once second strand synthesis has occurred. In the latter case, the genome can be detected regardless of where in the transduction pathway the genome is. This protocol can be combined with RNA FISH detection of reporter transcripts or endogenous transcripts, enabling measurement of expression and cell type classification.