TITLE: Metagenomic extraction of high molecular weight plankton from filters
AUTHORS: Benoît   Vacherie, Karine  Labadie

[DESCRIPTION]
Filtration and extraction protocol of plankton metagenomic samples to obtain high molecular weight DNA for sequencing on Minion nanopore.

The protocol describes the following steps:
- filtration of the water on filters of different porosity
- Recovery and grinding of organisms
- HMW extraction
- Results

[GUIDELINES]
Use only wide-bore pipette tips

[STEPS]
SECTION: Filtration
1. From several liters of water (fresh or sea).
Cascade filtration on filters of different porosity with the help of a perilstatic pump.

 

SECTION: Filtration
2. Pre-filtration through a 100µm celular sieve to remove large debris and larger zooplanktonic organisms (not of interest in our case).

 

SECTION: Filtration
3. Successive filtration on 3 filters of different porosities: 20 µm; 3 µm; 0.2 µm.
  
  
  

SECTION: Filtration
4. Filters are transferred into 5 ml tubes and flash frozen in liquid nitrogen.
The tubes are stored at -80°C before extraction.
  
Filters can be directly resuspended in nucléoprotect RNA  and stored at 4°C.
  

25. 
SECTION: Recovery and grinding of organisms
5. Add 3 ml of 1X PBS to the tube with the filter.
Vortex at maximum power to loosen the organisms from the membrane.
Remove the filter with sterile forceps.
  


SECTION: Recovery and grinding of organisms
6. Centrifuge the tube to pellet the organisms.
5000 x g, 5 min 
Gently remove the supernatant with a pipette.
SECTION: Recovery and grinding of organisms
6.1. If the filters have been stored in Nucleoprotect, make a second wash in 500 µl of 1X PBS (same centrifugation)
SECTION: Recovery and grinding of organisms
7. Resuspend the pellet with 800 µl of CD1 buffer (PowerSoil kit).
  

SECTION: Recovery and grinding of organisms
8. Incubate 10 min at 65 °C with 300 rpm agitation.
   


SECTION: Recovery and grinding of organisms
9. Transfer the suspension to a powerbeads tube (PowerSoil kit).
Vortex at maximum speed for 3 x 1 min (with 1 min pause between each grinding cycle) using the horizontal tube adapter.
3 min 

 

 

SECTION: Recovery and grinding of organisms
10. Transfer the grinding material by dipping the tip at the bottom of the tube into a 2ml tube.
SECTION: Powersoil extraction
11. Add 200 µl of CD2 solution (stored at 4°C) to the grinding and vortex 5 sec.
Centrifuge 1 min / 14 000 G / RT
14000 x g, 1 min 

SECTION: Powersoil extraction
12. Transfer the supernatant (avoiding touching the pellet) to a 2ml tube.
Add 600 µl of CD3 solution and vortex 5 sec.
SECTION: Powersoil extraction
13. Load 700 µl of the lysate on a MB spin column (PowerSoil kit)
Centrifuge 1 min / 14 000 G
14000 x g, 1 min 

SECTION: Powersoil extraction
14. Discard the filtrate and repeat step 13  until the lysate is consumed
SECTION: Powersoil extraction
15. Place the column on a new collection tube and add 500 µl of EA Solution
Centrifuge 1 min / 14,000 G
14000 x g, 1 min 

SECTION: Powersoil extraction
16. Discard the filtrate and add 500 µl of Solution C5
Centrifuge 1 min / 14,000 G
14000 x g, 1 min 

SECTION: Powersoil extraction
17. Place the column on a new collection tube
Centrifuge for 2 min / 14 000 G
14000 x g, 2 min 

SECTION: Powersoil extraction
18. Place the column on a 1.5 ml eppendorf tube
Add 25 µl of Solution C6 to the center of the column
Incubate 5 min at RT and Centrifuge 1 min / 14 000 G
5 min Room temperature 
14000 x g, 1 min 

SECTION: Powersoil extraction
19. Add 25 µl of Solution C6 to the center of the column
Incubate 5 min at RT and Centrifuge 1 min / 14,000 G 
Remove the column. 
5 min Room temperature 
14000 x g, 1 min 

SECTION: Powersoil extraction
20. Make the following quality controls:
- Qbit  (concentration)
- Nanodrop (purity)
- Tapestation or Femto-pulse (molecule size estimation)

Store the tube at -20°C
SECTION: Results
21.