TITLE: Coastal Environmental DNA Sampling &amp; Gravity Filtration Protocol
AUTHORS: Meghan M. Shea, Alexandria B Boehm

[DESCRIPTION]
This is a protocol for collecting coastal environmental DNA (eDNA) samples and gravity filtering them on site, using a set-up with single-use enteral feeding pouches first described in Curd et al. 2019 (see Appendix S6) and further elaborated in Gold et al. 2021. 


[STEPS]
SECTION: Pre-Sampling Preparation
3. Sterilize luer lock to hose barb adapters, male-female luer lock caps, and silicone rubber cap
SECTION: Pre-Sampling Preparation
1. Clean 1000 mL Nalgene bottles (as many as field blanks needed) with a 10% bleach rinse (leave for 10 minutes), then three rinses of DI water
SECTION: Pre-Sampling Preparation
2. Fill cleaned Nalgene bottles with 1000 mL of MilliQ water to use as field blanks 
SECTION: Pre-Sampling Preparation
3.1. Fill a container (e.g. old pipette tip boxes, so you can easily drain the water with the lid) with 10% bleach solution, add adapters and caps, and let sit for 10 minutes

  

SECTION: Pre-Sampling Preparation
3.2. Rinse adapters and caps 3 times with DI water

  

SECTION: Pre-Sampling Preparation
3.3. Place adapters and caps in a single layer in UV Crosslinker for 15 minutes
SECTION: Pre-Sampling Preparation
3.4. Store sterilized caps and adapters in sterile containers
SECTION: Pre-Sampling Preparation
4. Wipe down plastic storage bins with RNase Away, and organize needed field supplies (wiping individual items with RNase Away when possible as well).

Sampling Infrastructure & Transport: clothes rack, bricks for stability, shade structure, beach cart
Tools: tube cutter, flathead screwdriver, lab marker, temperature and salinity probe (e.g. Orion Model 1230 meter), prepared field blanks
Sampling Materials (1/sample): zip ties, 1000 mL Covidien single-use enteral feeding pouches, sterile 0.22 μm Sterivex filters, male luer lock to hose barb adapters, dual male-female luer lock caps, round silicone rubber caps, sterile 3 mL luer lock syringes, small bungees, whirl-pak bags
Preservation Materials: insulated cooler, ice packs

 

SECTION: Sampling Set-Up
5. Wheel cart with supplies to sampling location

  

6. Put together clothes rack for hanging enteral feeding pouches for gravity filtration and weigh down with bricks. Set up shade structure to cover clothes rack.
 
 
 

SECTION: Sampling
7. Put on fresh gloves 

SECTION: Sampling
8. Remove enteral feeding pouch from plastic wrapper

  

SECTION: Sampling
9. Label pouch with unique sample site/time identifier
SECTION: Sampling
10. Walk pouch to designated sampling site, rinse pouch three times with sampling water, and directly fill pouch with water to 1000 mL mark while keeping the tubing out of the water

 
SECTION: Sampling
11. Using temperature/salinity probe, record the temperature and salinity at the site the sample was collected
SECTION: Sampling
12. Secure the top of the pouch and return to clothes rack
SECTION: Sampling
13. Using a zip tie, secure the pouch to the top of the clothes rack

 
 
  

SECTION: Sampling
14. Using a small tube cutter, cut the tubing attached to the pouch at the end of the less-rigid opaque section beneath the first purple joint

  

SECTION: Sampling
15. Put on fresh gloves, and grab one luer lock adapter and a Sterivex filter
SECTION: Sampling
16. Place the barb end of the luer lock adapter into the cut end of the tubing, remove the Sterivex filter, and lock the filter to the tubing
SECTION: Sampling
17. Label the filter with an ethanol-proof lab marker with a unique sample site/time identifier
SECTION: Sampling
18. Using a small bungee cord, secure the filter to the bottom of the clothes rack so that it does not blow in the wind or brush against other filters

  

SECTION: Sampling
19. Using a flathead screwdriver, twist the inside of the purple connector on the pouch tubing to start the flow of water

   

SECTION: Sampling
20. Allow water to filter until all water has passed through (1-2 hours), or until drips slow to less than one/minute (clog); if the latter, record the amount of water that has passed through the filter
SECTION: Sampling
21. Remove the filter, and using a sterile 3 mL syringe, pass air through the filter until excess water has been removed
SECTION: Sampling
22. Cap the inlet end of the Sterivex filter with a male-female luer lock cap and the outlet end with a silicone rubber cap 
SECTION: Sampling
23. Label a small Whirl-Pak bag with the unique sample site/time and place the filter inside
SECTION: Sampling
24. Place bag on ice until transporting to -15°C freezer at the conclusion of field sampling
SECTION: Wrap-Up
25.1. Using a flathead screwdriver, twist the inside of the purple connector to stop the flow of water on any still-filtering bags
SECTION: Wrap-Up
25.2. Carefully lift the clothes rack on top of the cart; secure with zip ties

  

SECTION: Wrap-Up
26. Transport filters to -15°C freezer to await extraction
SECTION: Wrap-Up
25. If pouches need to be transported while still filtering:
SECTION: Wrap-Up
25.3. Using a flathead screwdriver, twist the inside of the purple connector to start the flow of water on any still-filtering bags. The cart can now be transported as pouches are still filtering.