TITLE: HMW DNA extraction for Long Read Sequencing using CTAB
AUTHORS: Benoît  Vacherie, Karine Labadie, Cyril Falentin

[DESCRIPTION]
High Molecular Weight DNA extraction protocol for Long Read sequencing.
Extraction is performed from flash frozen leaves stored at -80°C.
The protocol is adapted for an extraction of 1g of leaves.
This protocol is based on the protocol provided by Oxford Nanopore Technologies, Oxford, UK (ONT), "High molecular weight gDNA extraction from plant leaves" provided by the ONT community in March 2019, with slighly modifications. 
This protocol involves a conventional CTAB extraction followed by purification using commercial Qiagen Genomic tips (QIAGEN, MD, USA). DNA fragment size selection is performed using Short Read Eliminator (Circulomics, MD, USA).
This protocol is particularly adapted for plant leaves, but also works with many other organisms (microalgae, insects...).

[GUIDELINES]
Use only wide bore tips.
Work in a chemical hood when using 2-mercaptoethanol.
Allow the DNA to resuspend for at least of 24 hours before proceeding with QC.


[STEPS]
SECTION: preparation of reagents
1. Extraction Buffer : Prepare 20 mL of extraction buffer (per 1g of leaves) in a 50 mL tube

 

SECTION: DNA Extraction
2. Add 50 µl of 2-Mercaptoethanol to 20 ml of extraction buffer and preheat to 65 °C (approx. 15 min).
20 mL 
50 µL    
65 °C 

SECTION: DNA Extraction
3. Put a mortar in ice.
Cool the mortar and pestle with liquid nitrogen until the bubbling stops.
  

SECTION: DNA Extraction
4. Grind 1g of frozen sample to a fine powder (approx. 2 min), without adding liquid nitrogen.

1 g 2 min 

        
SECTION: DNA Extraction
5. Transfer the powder to the pre-warmed extraction buffer
SECTION: DNA Extraction
6. Add 40 µl of RNase A (100mg/ml) and vortex the tube 5 sec.
40 µL   
5 s 

SECTION: DNA Extraction
7. Incubate 1h at 65°C with intermittent agitation (300 rpm every 10 min)
60 min 

  


SECTION: DNA Extraction
8. Let the tube cool for 5 min at RT
5 min 

SECTION: DNA Extraction
9. Add 1 volume (20ml) of chloroform and vortex 2x5sec.
20 mL   

SECTION: DNA Extraction
10. Centrifuge with low acceleration and deceleration
5500 x g, 10 min, 4 °C 
  
       

SECTION: DNA Extraction
11. Gently transfer the aqueous phase to a new 50 ml tube.
SECTION: DNA Extraction
12. Add 0.7 volume of isopropanol and mix gently by inversion 10 times
  

SECTION: DNA Extraction
13. Put the tube at -80°C for 15 minutes.
15 min 

SECTION: DNA Extraction
14. If a DNA medusa appears after this step, recover the medusa using a Pasteur pipette (without breaking the tip of the pipette).
Wash the medusa in 3 successive baths of 70% ethanol then resuspend the medusa in 100µl of 1X TE.
Incubate for 1 hour at 55°C then store the tube at 4°C before quality control.
Continue the protocol with the rest of the tube (without the medusa).
If no  DNA medusa is visible continue the protocol.
SECTION: DNA Extraction
15. Centrifuge the tube with low acceleration and deceleration
5500 x g, 30 min, 4 °C 
SECTION: DNA Extraction
16. Carrefully remove the supernatant without resuspending the pellet.
Remove the remaining liquid by turning the tube upside down on a paper towel (make sure the pellet does not come off).
SECTION: DNA Extraction
17. Gently resuspend with the pipette the DNA pellet with 9.5 ml of G2 buffer (QIAGEN Genomic-tip 100/G).
  

SECTION: DNA Extraction
18. Incubate 15 min at 50°C. The sample can be stored overnight at 4°C at this stage.
15 min 

SECTION: Génomic tip purification
19. Equilibrate a QIAGEN Genomic-tip 100/G column with 4 ml of QBT buffer.
  
  

SECTION: Génomic tip purification
21. Allow all the QBT buffer to drain by gravity flow into a 50 ml tube.
SECTION: Génomic tip purification
22. Add the sample in G2 buffer (9.5ml) on the column and let it to enter the resin by gravity flow.
SECTION: Génomic tip purification
23. Wash the column twice with 7.5 ml of QC buffer.
  

SECTION: Génomic tip purification
24. Put the column on a 15 ml tube.
SECTION: Génomic tip purification
25. Elute the DNA with 5 ml of QF buffer preheated to 55°C.
  

SECTION: Génomic tip purification
20. Preheat 5 ml of QF buffer to 55°C.
  

SECTION: DNA précipitation
26. Add 0.7 volume of isopropanol to the eluate. 
Mix gently by inverting 20 times.
  

SECTION: DNA précipitation
27. Incubate 15 minutes at RT.
15 min 

SECTION: DNA précipitation
28. Centrifuge with low acceleration and deceleration
5500 x g, 30 min, 4 °C 
SECTION: DNA précipitation
29. Carefully discard the supernatant, gently resuspend the pellet with 1 ml of cold 70% ethanol.
  

SECTION: DNA précipitation
30. Transfer the resuspended DNA to a 1.5 ml DNA LoBind tube.

  

SECTION: DNA précipitation
31. Centrifuge with low acceleration and deceleration
5000 x g, 10 min, 4 °C 
SECTION: DNA précipitation
32. Carefully discard the supernatant.
Air dry the pellet at RT for about 10 minutes.
10 min 

SECTION: DNA précipitation
33. Resuspend the pellet with 50-100 µl of 1X TE buffer.
  

SECTION: DNA précipitation
34. Allow the DNA to resuspend for 2 hours at 55°C or ON at RT.
120 min  or   

SECTION: DNA précipitation
35. Store the DNA at 4°C.
SECTION: Sample quality control
36. Quantify your sample with a Qubit DNA HS assay.
Check the purity of the sample with a Nanodrop (measurements of 260/280 and 260/230 absorbance ratios).
Estimate the molecular weight of the sample with a Tapestation and/or a Femto pulse and/or a Pippin Pulse.
SECTION: Results
38.  QC results obtained on different  species.

    

SECTION: Sample quality control
37. Depending on the DNA concentrations and DNA length profiles, deplete short DNA molecules using SRE size selection kits (SRE XS, SRE or SRE XL kits).