Fusionfx device

For the first assay (Fig. 6A), 10⁸ vg of indicated AAV vectors were adjusted to the “pH buffer” (citric acid/Na₂HPO₄) at pH 7.2 and pH 5.2, for 15 min. at room temperature. Then samples were exposed to indicated temperatures (50, 55, 60, 65, 70, 75, 80 °C) for 15 min and then diluted in PBS. For the second assay (Fig. 6B), wells of a qPCR plate were loaded with 5 × 10⁸ vg of indicated AAV vectors diluted in PBS. The temperature gradient was generated by a LightCycler® 96 System (Roche Life Science) using a self-designed program (Supplementary Table S3). After the run, PBS was used to dilute samples. In both experiments, after PBS dilution, samples are transferred to a nitrocellulose membrane using a vacuum blotter for a native dot blot assay. After saturation, the membranes were incubated overnight at 4 °C with A20, or B1 antibodies^{63 (link)}. A horseradish peroxidase-conjugated anti-mouse antibody (Sigma,1/10,000 dilution) was then applied for 1 h at room temperature. Finally, the membranes were incubated with an enhanced chemiluminescence reagent (West Dura; Pierce) and analyzed by autoradiography film exposure or FusionFX device (Peqlab).

A 96‐well qPCR plate was loaded with 2E9 vector particles/well diluted in PBS and subjected to a temperature gradient using the LightCycler 96 System (Roche Life Science) as previously described (Rossi et al, 2019). Subsequently, samples were diluted in PBS and transferred to a nitrocellulose membrane using a vacuum blotter. Membrane was probed with B1 (1:5,000, 16048, Abcam) antibody recognising the C’‐terminus of all AAV2 capsid proteins and with A20 (1:50) antibody (Wistuba et al, 1997) binding to intact capsids. B1 and A20 were kindly provided by Martin Müller (DKFZ, Heidelberg, Germany). As secondary antibody, a horseradish peroxidase‐conjugated anti‐mouse antibody (1:10,000, Sigma‐Aldrich) was applied. Finally, the membranes were treated with an enhanced chemiluminescence reagent (West Dura, Pierce) and analysed by FusionFX device (Peqlab).