TITLE: isolation and extraction of plant nuclei in plug
AUTHORS: Karine  Labadie, Benoît   Vacherie

[DESCRIPTION]
Method for isolation and extraction of plant cell nuclei.
Protocol for obtaining UHMW DNA (> 150kb) allowing the production of optical cards with Bionano technology.

[GUIDELINES]
Never use a vortex in order to maintain a good molecule size.
Use only wide bore tips.
Allow the DNA to resuspend for a minimum of 48 hours before proceeding with QC.
The nuclei isolation part should be done in a chemical hood because of the use of 2-mercaptoethanol

[STEPS]
SECTION: preparation of reagents
1. NIB Buffer : 200 ml :   freshly prepared
 
Adjust the Ph to 9.4 then filter at 0.22 µm

SECTION: preparation of reagents
2. NIBT Buffer : 160 ml
 

SECTION: preparation of reagents
3. NIBTM Buffer : 40 ml
 

SECTION: preparation of reagents
5. Lysis Buffer : Can be stored for 1 year at RT.
 

SECTION: Nuclei isolation
6. Putting a mortar in ice
Cool a mortar/pestle with liquid nitrogen until the bubbling stops.
SECTION: Nuclei isolation
8. Grind 2g of frozen sample for  without adding liquid nitrogen, until a fine powder is obtained (approx. 2 min)
2 g 2 min 
    

SECTION: Nuclei isolation
7. Place a beaker in ice and add a magnetic stirrer.
Add 20 ml of NIBTM (10 ml/g of leaves) and stir gently
20 mL 

SECTION: Nuclei isolation
9. Transfer the powder to the beaker and shake gently for 10 minutes in ice.
10 min 
  
SECTION: preparation of reagents
4. Cell suspension Buffer : Can be stored for 1 year at 4°C.
 

SECTION: Nuclei isolation
10. Filter the mixture into a 50ml tube through autoclaved filters (2 cheese cloth + 2 Mira cloth) on a funnel (squeeze the filters at the end of filtration to recover more of the solution containing the nuclei).
Filter through a 40µm cell stariner into a new 50ml tube.
    
  

SECTION: Nuclei isolation
11. Pelleting the homogenate by centrifugation.(acceleration and deceleration at level 3)
800 x g, 20 min, 4 °C 
  
SECTION: Nuclei isolation
12. Remove the supernatant and gently resuspend the pellet in ice (use a brush if the pellet does not recover).
Add 20ml of cold NIBTM
20 mL 

SECTION: Nuclei isolation
13. Pelleting the homogenate by centrifugation to remove residues and unlysed cells.
60 x g, 2 min, 4 °C 
  
SECTION: Nuclei isolation
14. Filter the supernatant through a 40 µm cell sieve into a new 50 ml tube.
SECTION: Nuclei isolation
15. Pellet the nuclei by centrifugation.
800 x g, 15 min, 4 °C 
  
SECTION: Nuclei isolation
16. Wash the pellet 3 times in NIBT buffer :
    - Remove the supernatant
    - Gently resuspend the pellet
    - Add 40 ml of cold NIBT buffer 40 mL 
    - Centrifuge 800 x g, 15 min, 4 °C 
      
SECTION: Nuclei isolation
17. Make a final wash in 30 ml of cold NIB buffer.
800 x g, 15 min, 4 °C 
  
SECTION: Nuclei isolation
18. Resuspend the last pellet in the residual buffer (approx. 200 µl) and transfer the homogenate to a 1.5 ml tube.
Centrifuge.
800 x g, 15 min, 4 °C 
      
SECTION: Nuclei isolation
47. 
SECTION: Nuclei isolation
19. Remove the supernatant with a pipette and resuspend the pellet in an appropriate volume of cell suspension buffer : 
60µl / plug
Adjust the number of plugs to be made according to the size of the pellet.
SECTION: Embedding in agarose
20.  
 Use Low Melting agarose for a final agarose concentration of 0.8 %
  
SECTION: Embedding in agarose
21. Put a CHEF Disposable Plug Molds on ice
  
SECTION: Embedding in agarose
22. Melt agarose at 70°C for 5 min then equilibrate at 43°C for 5 min
5 min 70 °C 
5 min 43 °C 
SECTION: Embedding in agarose
23. Preheat the nuclei suspension to 43°C for 3min and then add the appropriate amount of 2% agarose (see table).
Mix gently with a wide-bore tip, avoiding bubbles.
3 min 43 °C 
SECTION: Embedding in agarose
24. Immediately dispense 100µl of mixture per well using wide-bore tips.
Allow to polymerise for 15 minutes on ice.
15 min on ice 

  
SECTION: Proteinase K digestion
25. Prepare a fresh proteinase K digestion solution by mixing 200 μl of proteinase K enzyme (20mg/ml) with 2.5 ml of  lysis buffer in a 50 ml tube.
200 µL  2.5 mL 

SECTION: Proteinase K digestion
26. Transfer plugs to the 50ml tube containing Proteinase K digestion solution.
SECTION: Proteinase K digestion
27. Incubate in thermomixer for 2 hours at 50 °C with intermittent mixing 
Mixing cycle: 10 seconds at 450 rpm followed by 10 minutes at 0 rpm
120 min 50 °C 

SECTION: Proteinase K digestion
28. Screw a sieve caps onto the tube and empty the solution. Change the proteinase K Solution bath as before.
Incubate in thermomixer overnight at 50 °C with intermittent mixing 
Mixing cycle: 10 seconds at 450 rpm followed by 10 minutes at 0 rpm
  50 °C 

SECTION: RNase Digestion
29. Prepare the wash solutions :

TE 10:50 (Wash Buffer)
  
TE 10:5 (For Rnase)
  

SECTION: RNase Digestion
30. Empty the tube using a vent cap.
  
Rinse the plugs 3 times with 10ml of wash buffer. 
Wash 2 times with 10ml wash buffer for 15 min at RT with gentle agitation (15 rpm) on a  horizontal platform mixer.
   

SECTION: RNase Digestion
31. Rinse the plugs 3 times with 10ml of TE 10:5
Add 2.5ml of TE 10:5 and 50 µl of Rnase Solution  
Incubate 1hour at 37°C with intermittent mixing
60 min 37 °C 

SECTION: RNase Digestion
32. Rinse the plugs 3 times with 10ml of Wash Buffer.
     NB : The plugs can be stored at 4°C in a wash buffer at this stage
SECTION: Agarase treatment
33. Wash 4 times with 10ml wash buffer for 15 min at RT with gentle agitation (15 rpm) on a  horizontal platform mixer.
  

SECTION: Agarase treatment
34. Transfer the plug to a 1.5 ml tube with a sterile spatula
SECTION: Agarase treatment
35. Melt the plug in a water bath at 70°C for 2 minutes
2 min 70 °C 

SECTION: Agarase treatment
36. Transfer the tube to a water bath at 43°C for 5 minutes
5 min 43 °C 

SECTION: Agarase treatment
37. Add 2µl of agarase and mix gently by rotating with the tip.
Incubate 45 minutes at 43 °C
45 min 43 °C 

SECTION: Dialysis
38. Place 10 ml of 1x TE Buffer in a 6 cm Petri dish.
Float a 0.1 μm dialysis membrane  on the surface of the 1x TE Buffer. Place a cover on the Petri dish and let the membrane equilibrates for 15 minutes.
15 min 

SECTION: Dialysis
39. Deposit the entire sample in the centre of the membrane using a wide-bore tip.
Place cover on the Petri dish and let the sample dialyze for 45 minutes at room temperature.
45 min Room temperature 

SECTION: Dialysis
40. Transfer DNA to a 1.5 ml microfuge tube with a Wide Bore Tip.
SECTION: Dialysis
41. Allow the DNA to resuspend overnight at RT then 2 days at 4°C before performing quality control.
  Room temperature 


SECTION: Sample QC
42. 
Quantify your sample with a Qubit HS. 
 NB : Before quantification, sonicate the DNA aliquot for 10 min to obtain a more reliable result

Visualise 1 µL of sample to estimate the molecular weight.  (Tapestation or/and pipin pulse or/and Femto pulse)
SECTION: Résults
43. QC results obtained on different plant species, using different technologies to estimate the size of the molecules.

   

SECTION: Nuclei isolation
19.1. optional : microscopic observation
    - Take an aliquot of 100 µl of suspension
    - Stain with DAPI
    - Observe the presence of nuclei