TITLE: Extraction and purification of viruses from stream biofilms
AUTHORS: Meriem Bekliz, Hannes Peter

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This series of sequential protocols can be used to extract and purify viruses from stream biofilm samples for metagenomic analysis. We compared each critical step of the protocol to alternatives and evaluated their performance using epifluorescence counting of viral-like particles and DNA yields. The pipeline consists of concentration of sample using tangential flow filtration, extraction using tetrasodium pyrophosphate and sonication and purfication using DNase I treatment, filtration, and ultracentrifugation in a sucrose density gradient. Where appropriate, we provide links to similar protocols.</div></div>

[STEPS]
?. [Before Starting]
Preparations
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							- 200 x TE Buffer: 2M Tris-HCl (pH 8.5) + 0.2M EDTA.- 1 x TE buffer: 10 mM Tris + 1 mM EDTA (pH 8.0).-  10 x DNase buffer: 100 mM Tris (pH 7.5) + 5mM CaCl2 + 25 mM MgCl2.
?. [Homogenization of biofilm sample]
Centrifuge biofilm sample at 100 x g for 15 minutes at 4°C.
?. [Concentration using TFF]
Use TFF to concentrate viruses following instruction posted here: https://www.protocols.io/view/Phage-Tangential-Flow-Filtration-c7yzpv/
?. [Enumerating VLPs using epifluorescence microscopy]
Compare to protocol: https://www.protocols.io/view/SYBR-Green-or-Gold-Staining-c7szndBecause of high background noise in biofilm samples, it is important that VLP are counted after purification and DNase I treatment.
?. [Nucleic acid extraction]
Add 0.1 volumes of 200 x TE buffer, 0.01 volumes of 0.5M EDTA (pH 8) and 1 volume of formamide to sample.Mix well and incubate at room temperature for 30 minutes.
?. [Purification using DNase I and sucrose gradient ultracentrifugation]
DNase I digestionAdd 0.1 volumes of DNase buffer 10 x.
?. [Extraction using tetrasodium pyrophosphate and sonication]
Add 5 mM (final concentration) of tetrasodium pyrophosphate to the concentrated biofilm sample.
?. [Purification using DNase I and sucrose gradient ultracentrifugation]
Sucrose gradient ultracentrifugationCompare to protocol posted here: https://www.protocols.io/view/Purifying-Viruses-Using-Sucrose-Cushion-c3wypd
?. [Homogenization of biofilm sample]
Recover the supernatant for downstream analyses.
?. [Extraction using tetrasodium pyrophosphate and sonication]
Incubate in dark for 15 minutes.
?. [Extraction using tetrasodium pyrophosphate and sonication]
Sonicate the sample on ice three times for 1 minute with 30 second intervals. Shake samples between sonication intervals.
?. [Extraction using tetrasodium pyrophosphate and sonication]
Centrifuge at 3234 x g for 15 minutes at 4°C.
?. [Extraction using tetrasodium pyrophosphate and sonication]
Filter sequentially the supernatant through 0.8 and 0.45 µm filter, respectively.
?. [Purification using DNase I and sucrose gradient ultracentrifugation]
Add 2.5 U/µL (final concentration) of DNase I enzyme.
?. [Purification using DNase I and sucrose gradient ultracentrifugation]
Incubate for 3 hours at 37˚C.
?. [Purification using DNase I and sucrose gradient ultracentrifugation]
Heat-inactivate DNase I enzyme at 65°C for 15 minutes.
?. [Purification using DNase I and sucrose gradient ultracentrifugation]
Check for microbial contamination with a standard PCR using universal 16S rRNA gene primers and gel electrophoresis.See for instance: https://www.protocols.io/view/16S-PCR-Universal-c5my45
?. [Purification using DNase I and sucrose gradient ultracentrifugation]
Pipette 3 mL of 66% sucrose and then 7 mL of 30% sucrose into the clean ultracentrifugation tube. Be careful not to mix the gradient.
?. [Purification using DNase I and sucrose gradient ultracentrifugation]
Mark layers with a permanent marker to facilitate layer recognition.
?. [Purification using DNase I and sucrose gradient ultracentrifugation]
Overlay the sample on top of the gradient and fill to the top. Be careful not to cause any mixing of the gradient.
?. [Purification using DNase I and sucrose gradient ultracentrifugation]
Ultracentrifuge at 106800 x g for 2 hours at 4°C. Use a slow acceleration and no brake for the deceleration.
?. [Purification using DNase I and sucrose gradient ultracentrifugation]
Using an 18 G needle, pierce carefully the tube between the 66% and 30% sucrose layers and extract approximately 2 mL.
?. [Enumerating VLPs using epifluorescence microscopy]
Stain sample with 2 µL 1000 x SYBR Gold and incubate for 30 minutes in the dark at room temperature.
?. [Enumerating VLPs using epifluorescence microscopy]
Carefully filter the sample onto 0.02 µm pore size membrane filter (Anodisc).
?. [Enumerating VLPs using epifluorescence microscopy]
Mount filter on glass slide using a drop of VectaShield.
?. [Enumerating VLPs using epifluorescence microscopy]
Screen the filter under fluorescence microscope using blue light excitation (488 nm) and green emission (512 nm).
?. [Nucleic acid extraction]
Add 2 volumes of 100% EtOH.
?. [Nucleic acid extraction]
Incubate at 4°C for 30 minutes.
?. [Nucleic acid extraction]
Centrifuge at 17000 x g for 20 minutes at 4°C to pellet the DNA.
?. [Nucleic acid extraction]
Resuspend DNA with 567 μL of 1 x TE buffer.
?. [Nucleic acid extraction]
Optional stopping point: store the sample at -20°C for up to one month.
?. [Nucleic acid extraction]
Pre-warm SDS at 37°C and CTAB at 65°C.
?. [Nucleic acid extraction]
Add 30 μL of 10% warm SDS and 3 μL of proteinase K (20 mg/mL).
?. [Nucleic acid extraction]
Mix and incubate for 1 hour at 37°C.
?. [Nucleic acid extraction]
Add 100 μL of 5M NaCl and mix.
?. [Nucleic acid extraction]
Add 80 μL of warm CTAB solution.
?. [Nucleic acid extraction]
Vortex and incubate at 65°C for 10 minutes.
?. [Nucleic acid extraction]
Add equal volume of chloroform.
?. [Nucleic acid extraction]
Vortex and centrifuge at 16000 x g for 10 minutes at room temperature.
?. [Nucleic acid extraction]
Transfer top aqueous layer to new microcentrifuge tube.
?. [Nucleic acid extraction]
Add equal volume of 25:24:1 phenol:chloroform:isoamyl alcohol.
?. [Nucleic acid extraction]
Vortex, and centrifuge at 16000 x g for 10 minutes at room temperature.
?. [Nucleic acid extraction]
Transfer top aqueous layer to new microcentrifuge tube.
?. [Nucleic acid extraction]
Add equal volume of chloroform to supernatant.
?. [Nucleic acid extraction]
Vortex, and centrifuge at 16000 x g for 10 minutes at room temperature.
?. [Nucleic acid extraction]
Transfer top aqueous layer to new microcentrifuge tube.
?. [Nucleic acid extraction]
Add 0.7 volumes of isopropanol to supernatant.
?. [Nucleic acid extraction]
Gently mix and incubate at -20°C overnight to precipitate DNA.
?. [Nucleic acid extraction]
Centrifuge at 16000 x g for 20 minutes at 4°C.
?. [Nucleic acid extraction]
Wash the DNA pellet twice with 70% cold EtOH.
?. [Nucleic acid extraction]
Resuspend DNA with 50 μL of DNase-free H2O.
?. [Nucleic acid extraction]
Quantify DNA (e.g. using Qubit according to manufacturer's instructions).
?. [Nucleic acid extraction]
Wash the pellet twice with 70% ice cold EtOH and allow to dry at room temperature.