TITLE: Virome harvesting from cheese microbiomes
AUTHORS: Adair  Borges

[DESCRIPTION]
This protocol details how we harvest the viral component, or virome, from cheese rind microbiomes. These viromes can be banked at 4 °C for short-term storage or -80 °C for long-term storage. Alternatively, one can extract DNA or RNA from these viromes for downstream sequencing or chemical analysis.

[STEPS]
SECTION: Virome extraction from cheese microbial community
2. Once the cheese sample is fully suspended, add 1 mL of chloroform to the sample. Close the conical tube, and seal the top with Parafilm. Shake the sample on a platform shaker for 1–2 h at room temperature.
SECTION: Virome extraction from cheese microbial community
3. Move the samples to a fresh 50 mL conical tube, as chloroform can weaken plastic. Next, spin the sample down at 10,000 x g for 10 min. Move the supernatant to a new conical tube, avoiding the pellet. Spin at 18,000 x g for 20 min. If the supernatant is clear, move to next step. If it is not, continue spinning at 18,000 x g for 10 min intervals. 
SECTION: Virome concentration
4. To concentrate the virome sample, we have used both PEG precipitation and a filter-concentrator-based protocol. The PEG protocol requires an overnight incubation, but the filter-concentrator generally requires more hands-on time. Use whichever phage concentration protocol works best for your circumstances.
SECTION: Virome extraction from cheese microbial community
1. Move a 0.5–1 g (chickpea-sized) sample of cheese rind to a 50 mL conical tube. Add 40 mL SM buffer (bioWORLD 41920012). Try to break up cheese rind by pipetting up and down. Vortexing could help, but don't be overly aggressive because you might destroy the phages.
SECTION: Virome concentration
5. To PEG-precipitate phage, first prepare 5× PEG precipitation solution containing 2.5 M NaCl and 20% w/v PEG8000.
 

SECTION: Virome concentration
6. Add PEG precipitation solution to the phage lysate to obtain a 1× concentration of 0.5 M NaCl and 4% w/v PEG8000. Mix by inverting the tube several times and refrigerate overnight at 4 °C.
5. Before concentrating, we recommend filtering the supernatant with a 0.22 µM Steriflip filter (EMD Millipore SCGP00525). This can help cut down on the carry-over of host nucleic acids into the phage prep. 
6. To concentrate the phage lysate, use a variation of the "Phage on Tap" method. After equilibrating a 100 kDa MWCO Amicon centrifugal filter unit (EMD Millipore UFC910024) with SM buffer (Bioworld 41920012-1), use the filter unit to concentrate the ~30 mL of phage lysate down to 0.5–1 mL. As the phage is being concentrated, use SM buffer to wash the sides of the filter.  
 

SECTION: Virome concentration
7. After the overnight incubation with PEG precipitation solution at 4 °C, pellet the PEG-precipitated phage particles by centrifuging at 19,000 × g for 60 min at 4 °C. A small white pellet should form at the bottom of the 50 mL conical tube.
SECTION: Virome concentration
8. Remove the supernatant, being careful not to disturb the PEG-precipitated phage pellet.
SECTION: Virome concentration
9. Resuspend the PEG-precipitated phage pellet in 300 µL SM buffer. This sample may be stored for the short term at 4 °C, or -80 °C for long-term storage. If storing at -80 °C, store in 20% glycerol with 1× PBS. You can also extract DNA or RNA directly from these samples.
7. This sample may be stored for the short term at 4 °C, or -80 °C for long-term storage. If storing at -80 °C, store in 20% glycerol with 1× PBS. You can also extract DNA or RNA directly from these samples.