Slide a lyzer 10 000 mwco dialysis cassette

Phage lysates were purified using cesium chloride gradient purification as described by ref. ^{69 (link)}. Briefly, 0.5–1 L of the crude lysates were treated with 5 mg/mL each of DNase I and RNAse (Roche) for 1 h at room temperature. The treated lysates were then mixed with NaCl to 1 M and solid PEG-8000 (Fischer) at 10% wt/vol and incubated at 4 °C overnight to precipitate the phage. The next day, the precipitation mixture was centrifuged at 3000 × g at 4 °C for 20 min and supernatant was discarded. The phage pellets were allowed to dry for 5–10 min before resuspension in 2–3 mL of SM buffer. Chloroform was then added at 1:3 of total volume and mixed for 2 min to remove residual PEG. The mixture was then centrifuged for 10 min at 12,000 × g at 4 °C and then the aqueous top layer was transferred to a 50 mL conical tube (Corning Falcon). SM buffer was added to bring the total volume up to 4.5 mL and mixed with 2.25 g of Cesium chloride (CsCl). CsCl density gradients were set up in a Beckman ultra-clear ultracentrifuge tube using 1.7, 1.5, and 1.45 g/mL layers, each at 2 mL, with the phage mixture added on top. The gradient was centrifuged at 60,000 × g at 4 °C for 3 h to produce a visible phage band which was extracted using a sterile 23 G needle. Any remaining CsCl was removed by dialysis using a Slide-a-Lyzer 10,000 MWCO dialysis cassette (Thermo Scientific) in SM buffer.

CrPV and ELVs were first separated via iodixanol gradients as described above. Fractions corresponding to either ELVs or CrPV virions were pooled and dialyzed in PBS using a Slide-A-Lyzer 10,000 MWCO dialysis cassette (Thermo Fisher). After dialysis, pooled fraction sets were added to S2 cells seeded in a 6-well plate without media and incubated for 1 h at 25 °C. Following incubation, cells were washed with PBS and complete S2 cell media was added. Cells were incubated at 25 °C for 48 h before being harvested for viral yield determination.