TITLE: Wisecaver Lab DNAzol-based extraction of high molecular weight DNA from photosynthetic sea slugs
AUTHORS: Jennifer H Wisecaver, Raeya Ogas

[DESCRIPTION]
Protocol for DNAzol-based extraction of high molecular weight DNA from photosynthetic sea slugs. This streamlined protocol takes less time compared to the lab's prior CTAB-based protocol while still yielding high quality of DNA for Illumina and Oxford Nanopore sequencing. 

[STEPS]
SECTION: Homogenization and DNA extraction
6. Add 300 µL DNAzol. Pipet up and down to mix and transfer to a 1.5 mL centrifuge tube. 

SECTION: Homogenization and DNA extraction
7. Add 5 µL RNAse A. Make sure the cap is closed securely, and mix well by vigorously shaking the tube for 15 s.

SECTION: Homogenization and DNA extraction
8. Incubate on the ThermoMixer at  . About half way through the incubation, take the tubes out and again mix well by vigorously shaking the tube for 15 s. Place back in the ThermoMixer to complete the incubation step.

SECTION: Homogenization and DNA extraction
9. Add 300 µL chloroform to each sample. Make sure the cap is closed securely, and mix well by vigorously shaking the tube for 15 s. 

All liquid chloroform liquid waste should be ejected or decanted into the amber bottle labeled 'Phenol:Chloroform Waste' inside the fume hood. Any plasticware that comes into contact with chloroform needs to be properly disposed of in the sharps bin inside the fume hood labeled 'Mutagen/Carcinogen Sharps Do Not Autoclave'.

SECTION: DNA Precipitation
13. Add 225 µL of 100% ethanol. Invert ten times to mix.
SECTION: DNA Precipitation
15. Centrifuge at 5000 rpm, 4 min. Orient the centrifuge tube so that cap hinge is on the outer edge of the rotor. 

Proper orientation of the tube helps to avoid the pelleted RNA when removing the supernatant in subsequent steps. Even if you can't see the pellet, you will know that it is located on the on the bottom corner under the hinge. 

SECTION: DNA Precipitation
17. Add 300 µL of previously prepared DNAzol wash and drag along the top of a tube rack to agitate the sample and dislodge the pellet.

SECTION: DNA Precipitation
18. Incubate at room temperature for 5 min.

SECTION: DNA Precipitation
21. Add 300 µL 75% ethanol wash and vortex briefly. If the pellet does not dislodge at this step, that is okay.
SECTION: DNA Precipitation
23. Discard the supernatant by taking off the initial volume with a 1 mL pipette tip. Briefly spin down the tube using the little bench top spinner, and then use a 200 mL pipet tip to get the last bit of liquid. 

Again, even if you can't see the pellet, you will know that it is located on the on the bottom corner under the hinge. So be careful to avoid that side of the tube. Pipette away the supernatant by tipping the tube slightly towards you and pipetting the liquid from the bottom corner opposite the hinge. 
SECTION: DNA Precipitation
25. Resuspend sample in 50 µL TE Buffer.

SECTION: DNA Precipitation
26. Incubate on the ThermoMixer  .

SECTION: DNA Precipitation
27. Briefly spin down the tube using the little bench top spinner.
SECTION: QC and clean up
30. While the TapeStation is running, measure the DNA concentration using the Qubit.
SECTION: Prepare Station and Materials
1. Set the ThermoMixer to 35 °C 

SECTION: Prepare slug sample(s)
4. Freeze slug tissue in liquid nitrogen and pulverize using a Tissue Lyser II (Qiagen) or a mortar and pestle 
SECTION: Prepare Station and Materials
2. Get liquid nitrogen.
SECTION: Prepare Station and Materials
3. Wipe down your work area with 70% ethanol:
Chemical fume hood
Benchtop
Rotors on the microcentrifuge and bench top spinner
Pipettes
SECTION: Prepare slug sample(s)
5. Immediately add the DNAzol (see step 9) or store in -80 °C freezer. 

It's important to work quickly here as the liquid nitrogen will have lysed many cells already. Material will be highly susceptible to environmental nucleases if allowed to sit at room temperature. 

SECTION: Homogenization and DNA extraction
10. Incubate on the Hula Mixer for 5 min 
SECTION: Homogenization and DNA extraction
11. Centrifuge at 12000 rpm, 10 min. Don't jostle samples when removing from the centrifuge.
SECTION: Homogenization and DNA extraction
12. Transfer approximately 300 µL of the top aqueous phase to a fresh 1.5 mL tube. Pipet slowly from the top, being careful not to disrupt the interphase. 
SECTION: DNA Precipitation
16. Discard the supernatant by taking off the initial volume with a 1 mL pipette tip. Use a 200 mL pipette tip to get the last bit of liquid. 

This is the last supernatant and plastic waste that needs to be disposed of in the phenol:chloroform waste. All waste in the remaining steps can go in the standard lab 'look-alike' waste bin. 
SECTION: DNA Precipitation
20. Discard the supernatant by taking off the initial volume with a 1 mL pipette tip. Use a 200 mL pipette tip to get the last bit of liquid. 

Be sure to avoid the hinge side of the tube where the sample is. 
SECTION: DNA Precipitation
22. Centrifuge at 5000 rpm, 4 min. Orient the centrifuge tube so that cap hinge is on the outer edge of the rotor. 

SECTION: DNA Precipitation
24. Air dry the DNA pellet for 5 min by placing the tube on its side inside a folded kim wipe. 

Do not over dry the DNA pellet as this will greatly decrease its solubility. However, also be sure that all the ethanol is removed as residual ethanol will also decrease solubility. 
SECTION: QC and clean up
28. Make sure all chloroform waste is properly disposed of in the chemical fume hood. 
Wipe down the chemical fume hood and the benchtop with 70-75% ethanol. 


SECTION: Homogenization and DNA extraction
8.1. During incubation, prepare an aliquot of 100% ethanol. Prepare 300 µL per sample.
SECTION: Homogenization and DNA extraction
8.2. During incubation, prepare an ethanol and DNAzol wash by mixing 1 mL DNAzol with 750 µL ethanol.
SECTION: Homogenization and DNA extraction
8.3. During incubation, prepare a 75% ethanol wash using nuclease free water. Prepare 300 µL per sample.
SECTION: DNA Precipitation
14. Incubate at room temperature for 5 min.
SECTION: DNA Precipitation
19. Centrifuge at 5000 rpm, 4 min. Orient the centrifuge tube so that cap hinge is on the outer edge of the rotor. 
SECTION: QC and clean up
29. Assess DNA integrity using the TapeStation. Make sure you reserve time on the TapeStation Teams calendar. 
SECTION: QC and clean up
31. If the TapeStation reports a high DNA integrity (DIN) score (≥ 7) and DNA concentration ≥ 20 ng/uL, then you have successfully isolated DNA for whole genome sequencing!
   
Cap your sample tube tightly. Make sure each tube is labeled on the cap and vertically on the side with the sample's unique identifier*. Wrap the cap with parafilm and place sample in the 'Novogene' box in the full sized -20 freezer.   

*The identifier should start with a letter and be no more than 12 characters long. Write it carefully using a fine tip permanent marker as this tube will be shipped off for DNA sequencing.