TITLE: Cryogrinding protocol : mecanic lysis of planktonic filter for RNA/DNA extraction 
AUTHORS: Sarah  Romac

[DESCRIPTION]
Cell Lysis can be difficult to conduct for envirronmental water samples collected on big filters, specially when big water volumes (20-100L) have been filtered on polycarbanate 142mm-diameter filters.
Here we developped a mecanic lyis protocol in liquid nitrogen conditions to get preserved DNA and RNA.
This protocol was developped for combined RNA and DNA extraction from environmental water samples for Biomarks (2009-2013) and TARA OCEANS projects (2009-ongoing).

[BEFORE_START]
- Wash all the area and Becher 1L with EtOH 70% and RNase away.
- Put 2 glove pairs to be more protected from the cold.
- Put each material (tweezers, cisors, spoon, Falcon 50, FreezerMill equipment, teethbrush) at least 20min under UV light before and between each cryogrinding.

[GUIDELINES]
Liquid nitrogen should be handled in well-ventilated areas.
Wear a labcoat, nitrile gloves and googles.
Put 2 glove pairs to be more protected from the cold.
Wear cold-specific gloves when you work in contact with liquid nitrogen.
To not degrade RNA, all work must be done with liquid nitrogen.


[STEPS]
SECTION: FreezerMill Tube Assembly
11. Finish the assembly of the cryogrinding tube : insert the magnet inside the tube and close the tube with the other extremity containing the hole.

  

SECTION: FreezerMill Tube Assembly
12. Put the tube in the FreezerMill chamber, and check the liquid nitrogen level and close the FreezerMill.
  

SECTION: FreezerMill Run Conditions
14. Set up the FreezerMill cryogrinding conditions : 

Precool: 2 min
Run time: 1 min

Cycles number: 1
Rate: 5 knocks per sec. (respect roughly this condition!).


SECTION: Powder Collection
20. Replace the Falcon50 in the liquid nitrogen in the Becher and process to the cryogrinding of the second part of the 142mm filter.

For that, take the Falcon50 containing the half-part left of the 142mm filter and repeat steps 9 to 18.
SECTION: Washing and Cleaning
22. Disassemble  the cryogrinding tubes.

Wash thoroughly all the parts of the cryogrinding tubes, and the FreezerMill extractor  using a teethbrush to remove powder residues following this order :
- 1% diluted bleach ;
- tap water ;
- milliQ water ;
- EtOH 70% .

Decontaminate the bench, and Becher using RNase away for next sample cryogrinding.

Put all the small equipment cited above (including the teethbrush) on an alumin paper and incubate them into UV-light at least 20min. (in a PCR hood or a cross-linker for instance, https://www.dutscher.com/frontoffice/product?produitId=0O-78-39)
SECTION: FreezerMill Preparation
3. Check the level using a 10mL pipette (maximum level of liquid ntrogen should correspond to 5 mL on pipette's graduation).




SECTION: FreezerMill Tube Assembly
6. Pour a half the 1 L Becher with liquid nitrogen.

Dive the sample tube containing the 142mm filter in the Becher to keep it deep-frozeen.

 

SECTION: FreezerMill Preparation
2. Close delicately the lid of the FreezerMill.


SECTION: FreezerMill Preparation
1. Fill the Freezer Mill tank : pour Liquid Ntrogen in the tank until the "up limit" graduation.


 

SECTION: FreezerMill Preparation
4. Wait for 20-30 min until the Freezer Mill reachs in temperature.

SECTION: FreezerMill Tube Assembly
7. Assemble the bases for 2 FreezerMill cryogrinding tubes :

  

SECTION: FreezerMill Tube Assembly
8. Put the 2 half-assembled tubes in the Becher .

   

SECTION: FreezerMill Tube Assembly
9. Take the sample tube with the tweezers, open the tube and take the filter with the tweezers.

If the filter resists, delicately break the tube using the crab tweezer keeping holding the tube with the tweezer.
SECTION: FreezerMill Tube Assembly
10. Take away the filter with the tweezer and cut the half of the filter using the cizors and put it in a Falcon50. Put this Falcon in the liquid Nitrogen.

Cut the other half-part filter again in twice and put these 2 pieces in one of the 2 frozen cryogrinding tube.

  


SECTION: FreezerMill Run Conditions
15. Start the run.
SECTION: FreezerMill Tube Assembly
13. Put a sterile Falcon50 correctly annotated (Sample code) in the Becher containing liquid Nitrogen for cooling.

This Falcon will be used to collect the final powder from the cryogrinded filter.
SECTION: Powder Collection
16. When the run is "complete", open delicately the FreezerMill and take the cryogrinding tube using the FreezerMill extractor.

  

 

SECTION: Powder Collection
17. Put the cryogrinding tube in the Becher containing liquid nitrogen.
SECTION: Powder Collection
18. Open delicately the cryogrinding tube using the FreezerMill extractor, and remove the magnet using tweezers.
SECTION: Powder Collection
19. Transfer the powder obtained in the Falcon50 tube by directly inversing the the cryogrinding tube inside the Falcon50. 

Afterthat, use the spatula to collect a maximum of powder. 

Recover most of material you can using the spatula.
SECTION: Powder Collection
21. At the end of the 2 cryogrinding runs, store the Falcon50 tube containing the powder at -80°C until RNA/DNA extraction process.
SECTION: FreezerMill Preparation
5. Choose the format of the filter for next steps :
SECTION: FreezerMill Tube Assembly
6. Pour a half the 1 L Becher with liquid nitrogen.


Dive the sample tube containing the 142mm filter in the Becher to keep it deep-frozeen.


 



SECTION: FreezerMill Tube Assembly
7. Assemble the bases for 1 FreezerMill cryogrinding tube :

  
SECTION: FreezerMill Tube Assembly
8. Put the half-assembled tube in the Becher .

   

SECTION: FreezerMill Tube Assembly
9. Take the sample tube with the tweezers, open the tube and take the filter with the tweezers.

If the filter resists, delicately break the tube using the crab tweezer keeping holding the tube with the tweezer.
SECTION: FreezerMill Tube Assembly
10. Take away the filter with the tweezer and put it in the frozen cryogrinding tube. 


SECTION: FreezerMill Tube Assembly
11. Finish the assembly of the cryogrinding tube : insert the magnet inside the tube and close the tube with the other extremity containing the hole.

  

SECTION: FreezerMill Tube Assembly
12. Put the tube in the FreezerMill chamber, and check the liquid nitrogen level and close the FreezerMill.
  

SECTION: FreezerMill Tube Assembly
13. Put a sterile Falcon50 correctly annotated (Sample code) in the Becher containing liquid Nitrogen for cooling.

This Falcon will be used to collect the final powder from the cryogrinded filter.
SECTION: FreezerMill Run Conditions
14. Set up the FreezerMill cryogrinding conditions : 

Precool: 2 min
Run time: 1 min

Cycles number: 1
Rate: 5 knocks per sec. (respect roughly this condition!).


SECTION: FreezerMill Run Conditions
15. Start the run.
SECTION: Powder collection
16. When the run is "complete", open delicately the FreezerMill and take the cryogrinding tube using the FreezerMill extractor.

  

 

SECTION: Powder collection
17. Put the cryogrinding tube in the Becher containing liquid nitrogen.
SECTION: Powder collection
18. Open delicately the cryogrinding tube using the FreezerMill extractor, and remove the magnet using tweezers.
SECTION: Powder collection
19. Transfer the powder obtained in the Falcon50 tube by directly inversing the the cryogrinding tube inside the Falcon50. 

Afterthat, use the spatula to collect a maximum of powder. 

Recover most of material you can using the spatula.
SECTION: Powder collection
20. Store the Falcon50 tube containing the powder at -80°C until RNA/DNA extraction process.
SECTION: Washing and Cleaning
21. Disassemble  the cryogrinding tubes.

Wash thoroughly all the parts of the cryogrinding tubes, and the FreezerMill extractor  using a teethbrush to remove powder residues following this order :
- 1% diluted bleach ;
- tap water ;
- milliQ water ;
- EtOH 70% .

Decontaminate the bench, and Becher using RNase away for next sample cryogrinding.

Put all the small equipment cited above (including the teethbrush) on an alumin paper and incubate them into UV-light at least 20min. (in a PCR hood or a cross-linker for instance, https://www.dutscher.com/frontoffice/product?produitId=0O-78-39)