TITLE: Liquid biopsy in sentinel mussels 
AUTHORS: Sophia Ferchiou, France Caza, Yves St-Pierre

[DESCRIPTION]
This protocol has been optimized for sampling hemolymph in sentinel mussels in remote areas, such as polar regions. A detailed protocol can be found in Caza et al. (2019).

[BEFORE_START]
A. Sampling Section (Protocol optimized for freshly collected mussels)
Be sure to gather all materials before collecting mussels. 

B. Extraction of circulating cell-free DNA Section
1) Equilibrate samples to room temperature (15–25°C).
2) Equilibrate Buffer ATE to room temperature.
3) Set a thermomixer or heated orbital incubator to 56°C for use in step #13.
4) Ensure that buffers AW1 and AW2 have been prepared :
    a) Add 25 mL ethanol (100%) to the bottle containing 19 mL Buffer AW1 concentrate.
    b) Add 30 mL ethanol (100%) to the bottle containing 13 mL Buffer AW2 concentrate.
5) If Buffer AL or Buffer ATL contains precipitates, dissolve by heating to 70°C with gentle agitation.
6) Reconstitute carrier RNA by adding 310 µL of buffer ATE. 

[STEPS]
SECTION: Sampling
1. Collect mussels (Mytilus spp.) with a shell length range between 50 to 70 mm. 
SECTION: Sampling
2. Remove intervalvar liquid by opening gently the valves with the tip of a knife.
SECTION: Sampling
4. Transfer immediately the hemolymph to a sterile 1.5 mL Eppendorf tube (Optional: You can pool 3 or 4 hemolymph samples to obtain a total volume of 1.5 mL per tube).
SECTION: Sampling
5. Centrifuge for 3 minutes at maximum speed (approx. 3000 xg) at room temperature using a battery-powered mini-centrifuge (TOMY, Japan) or equivalent.
SECTION: Sampling
6. After centrifugation, transfer the supernatant (clear solution above pellet at bottom of tube) in a new 1.5 mL Eppendorf tube and gently resuspend the pellet. The pellets can be frozen (-20°C) or spotted on FTA cards until used use. Supernatants can also be frozen (-20°C) or spotted on FTA cards (using 70 µL aliquots) until use. We routinely use  Whatman 903™FTA cards with 13 mm discs to spot pellets or supernatants.

Note : Once spotted, FTA cards are dried for 15 minutes at room temperature and stored individually in small ziplock plastic bags containing one silica gel desiccant moisture absorber.

Note : Unless otherwise indicated, hemolymph should be collected and processed within one hour after sampling.
SECTION: Extraction of circulating cell-free DNA
8. Unless otherwise indicated, you can use the QIAamp DNA Investigator Kit (Qiagen) for extraction of the hemolymphatic circulating cell-free DNA.
SECTION: Extraction of circulating cell-free DNA
9. First, thaw frozen supernatants at room temperature and clarify by centrifugation for 10 minutes at 4500 xg at room temperature. 
SECTION: Extraction of circulating cell-free DNA
10. Transfer 70 μL of the clarified supernatant into a new sterile 1.5 mL Eppendorf tube. 
SECTION: Extraction of circulating cell-free DNA
12. Add 100 μL of buffer AL and 1 µL of reconstituted RNA Carrier (1µg/µL). Mix by pulse-vortexing for 15 seconds or until it is a homogeneous solution.
SECTION: Extraction of circulating cell-free DNA
11. Add 30 µL of buffer ATL and 10 μL of proteinase K (>600 mAU/mL) to the sample.
SECTION: Extraction of circulating cell-free DNA
13. Incubate at 56°C for 10 minutes on a lab orbital shaker set at 900 rpm.
SECTION: Extraction of circulating cell-free DNA
14. Add 50 μL of ethanol (100%) and mix thoroughly by pulse-vortexing for 15 seconds. Then, incubate for 3 minutes at room temperature.
SECTION: Extraction of circulating cell-free DNA
15. Transfer the entire lysate from step #14 to the QIAamp MinElute column. 
SECTION: Extraction of circulating cell-free DNA
16. Centrifuge at 6000 x g for 1 minute. Place the QIAamp MinElute column in a clean 2 mL collection
tube and discard the collection tube containing the flow-through.
SECTION: Extraction of circulating cell-free DNA
17. Add 500 μL of buffer AW1 to the QIAamp MinElute column. 
SECTION: Extraction of circulating cell-free DNA
18. Repeat Step #16.
SECTION: Extraction of circulating cell-free DNA
19. Add 700 μL of buffer AW2 to the QIAamp MinElute column. 
SECTION: Extraction of circulating cell-free DNA
20. Repeat Step #16.
SECTION: Extraction of circulating cell-free DNA
21. Add 700 μL of ethanol (100%) to the QIAamp MinElute column. 
SECTION: Extraction of circulating cell-free DNA
22. Repeat Step #16.
SECTION: Extraction of circulating cell-free DNA
23. Centrifuge at full speed (20,000 x g) for 3 minutes at room temperature.
SECTION: Extraction of circulating cell-free DNA
24. Place the QIAamp MinElute column in a clean 1.5 mL Eppendorf tube and discard the collection tube containing the flow-through.
SECTION: Extraction of circulating cell-free DNA
25. Open the lid and incubate at room temperature for 10 minutes.
SECTION: Extraction of circulating cell-free DNA
26. Apply 60 μL of buffer ATE to the center of the membrane.
SECTION: Extraction of circulating cell-free DNA
27. Close the lid and incubate at room temperature for 10 minutes.
SECTION: Extraction of circulating cell-free DNA
28. Centrifuge at full speed (20,000 x g) for 1 minute.
SECTION: Extraction of circulating cell-free DNA
29. Discard the QIAamp MinElute column and keep the eluate. 
SECTION: Sampling
3. Withdraw hemolymph from the adductor muscle using a syringe fitted with a 25G needle.
SECTION: Sampling
7. Bring the mussels back to their respective mussel bed.