Recombinant AAVs were generated by triple transfection of 293T cells (ATCC) using polyethylenimine (PEI)46 . Viral particles were harvested from the media at 72 hrs post transfection and from the cells and media at 120 hrs. Cell pellets were resuspended in 10mM Tris with 2mM MgCl2, pH 8, freeze-thawed three times, and treated with 100 U/mL Benzonase (Epicentre) at 37°C for at least 1 hr. Viral media was concentrated by precipitation with 8% polyethylene glycol 8000 (Sigma-Aldrich) with 500 mM sodium chloride47 (link), resuspended in Tris-MgCl2, and then added to the lysates. The combined stocks were then adjusted to 500 mM NaCl, incubated at 37°C for 30 minutes, and clarified by centrifugation at 2000 × g. The clarified stocks were then purified over iodixanol (Optiprep, Sigma; D1556) step gradients (15%, 25%, 40% and 60%)48 (link). Viruses were concentrated and formulated in phosphate buffered saline (PBS). Virus titers were determined by measuring the number of DNaseI-resistant vg using qPCR with linearized genome plasmid as a standard46 .
For capsid library virus generation, two modifications were made to the above virus production protocol to reduce the production of mosaic capsids that could arise from the presence of multiple capsid sequences in the same cell. First, only 10 ng of the rAAV-Cap-in-cis library plasmid was transfected (per 150 mm dish) to increase the likelihood that most transfected cells only received one capsid variant sequence. Second, the virus was collected at 48 hrs (media) and 60 hrs (cells and media), rather than at 72 hrs and 120 hrs as described above, to minimize the secondary transduction of producer cells with rAAV library virus released into the medium.
For capsid library virus generation, two modifications were made to the above virus production protocol to reduce the production of mosaic capsids that could arise from the presence of multiple capsid sequences in the same cell. First, only 10 ng of the rAAV-Cap-in-cis library plasmid was transfected (per 150 mm dish) to increase the likelihood that most transfected cells only received one capsid variant sequence. Second, the virus was collected at 48 hrs (media) and 60 hrs (cells and media), rather than at 72 hrs and 120 hrs as described above, to minimize the secondary transduction of producer cells with rAAV library virus released into the medium.
