TITLE: High molecular weight DNA extraction for marine macroalgal tissue
AUTHORS: Malia Moore, Taylor S. Steele

[DESCRIPTION]
This protocol details high molecular weight DNA extraction for marine macroalgal tissue. Marine macroalgae contain a variety of unique cell wall components including sulfated polysaccharides and polyphenolics. These components often co-elute with high molecular weight (HMW) DNA and lead to reduced library prep and sequencing outcomes. This protocol incorporates polyvinylpolypyrrolidone (PVPP) and β-mercaptoethanol (BME) to reduce polyphenolic contamination, and an early salting out step with potassium acetate (KOAc) to address polysaccharides. This protocol is largely adapted from an Oxford Nanopore HMW DNA extraction from Arabidopsis leaves, which incorporates the QIAGEN Blood and Cell Culture DNA Midi Kit for column cleanup. The DNA product often requires additional cleanup after elution, and we suggest the BluePippin 15kb size selection for all HMW applications.

[GUIDELINES]
Marine macroalgae contain a variety of unique cell wall components including sulfated polysaccharides and polyphenolics. These components often co-elute with high molecular weight (HMW) DNA and lead to reduced library prep and sequencing outcomes. This protocol incorporates polyvinylpolypyrrolidone (PVPP) and β-mercaptoethanol (BME) to reduce polyphenolic contamination, and an early salting out step with potassium acetate (KOAc) to address polysaccharides.1 This protocol is largely adapted from an Oxford Nanopore HMW DNA extraction from Arabidopsis leaves, which incorporates the QIAGEN Blood and Cell Culture DNA Midi Kit for column cleanup.2 The DNA product often requires additional cleanup after elution, and we suggest the BluePippin 15kb size selection for all HMW applications.

Additional tips:

In the field or in lab, it is vital to scrape off all surface epiphytes and wash the sample in clean water before flash freezing to reduce contaminants common in the marine environment that confound genome assembly.
Marine macroalgae are incredibly diverse in biochemical content, so individual seaweeds may require troubleshooting. Suggested alterations include varying input tissue type or quantity, increasing CTAB or BME percent, or adding a second chloroform separation.
It may be necessary to carry out extractions of the same tissue in parallel to yield sufficient DNA, especially when large losses from BluePippin are expected. It is not suggested to combine multiple extractions onto the same column, as this may lead to overloading and a dirty sample. This protocol as written, paired with BluePippin, has produced sequencing-quality DNA for Nanopore from a red alga Porteria hornemanii and a brown alga Macrocystis pyrifera. For P. hornemanii, a single 20 mL extraction produced sufficient DNA for sequencing, but for M. pyrifera, three parallel extractions of 20 mL were necessary.


[STEPS]
SECTION: Lyophilizing algal tissue
1. Flash-freeze algal tissue in liquid nitrogen (target ≥5 g wet tissue).

SECTION: Setting up the DNA extraction
5. Prepare desired volume of Carlson lysis buffer (100 millimolar (mM) Tris-HCl, pH 9.5, 2% CTAB, 1.4 Molarity (M) NaCl, 1% PEG 8000, 20 millimolar (mM) EDTA) and mix 1440 min on a magnetic stirrer. The stock solutions suggested under consumables will yield a homogenous buffer with no precipitate.
SECTION: Lyophilizing algal tissue
2. Quickly transfer sample to lyophilization container and freeze dry for 36-48 hours.
SECTION: Lyophilizing algal tissue
3. Macerate the tissue with a clean spatula to increase surface area and put on the lyophilizer for another 1440 min.

SECTION: Lyophilizing algal tissue
4. Remove and refrigerate with desiccant for immediate use, or store at -80 °C for longer periods.

SECTION: Setting up the DNA extraction
6. Pre-heat a heat block or water bath to 65 °C and place in a fume hood.

SECTION: Setting up the DNA extraction
7. For each extraction, transfer 20 mL of Carlson lysis buffer to a 50-ml Falcon tube.

SECTION: Setting up the DNA extraction
8. In a fume hood, add 400 µL BME (originally 50 µL) and mix by vortexing. Pre-warm the solution to 65 °C for 30 min before starting the extraction.

SECTION: Setting up the DNA extraction
9. Scoop 0.5 teaspoons lyophilized plant tissue into a clean mortar and add 50-100 mg powdered PVPP. Grind with pestle for ~30 s, until tissue is powdered and combined, but not long enough to introduce significant moisture. Move immediately into DNA extraction.
SECTION: Lysis and first precipitation
10. Pour tissue into the warm buffer. Invert 5 times. 
SECTION: Lysis and first precipitation
11. Add 40 µL of RNase A and vortex for 5 s.

SECTION: Lysis and first precipitation
12. Optional: If using a heat block with mixing, set the block (still at 65 °C) to mixing at  .
SECTION: Lysis and first precipitation
13. Incubate for 60 min at 65 °C. 

SECTION: Lysis and first precipitation
14. Allow the tubes to cool down to Room temperature for 10 min.

SECTION: Lysis and first precipitation
15. Add 20 mL chloroform and vortex for two pulses of 5 s each.

SECTION: Lysis and first precipitation
16. Centrifuge the tubes at 3500 x g, 15 min, 4 °C.

SECTION: Lysis and first precipitation
17. In a fume hood, transfer the top layer of lysate from each tube to a new 50-ml Falcon tube, without disturbing the interphase. 
 

SECTION: Lysis and first precipitation
18. Mix supernatant with 0.4X 5 Molarity (M) potassium acetate (KOAc) at Room temperature, inverting at least 10 times to combine, then incubate on ice for 20 min.

SECTION: Lysis and first precipitation
19. Centrifuge the tubes at 3500 x g, 45 min, 4 °C.
SECTION: Lysis and first precipitation
20. Remove and retain the supernatant. 
 

SECTION: Lysis and first precipitation
21. Add 0.7X volumes of isopropanol. Invert 10 times. Incubate at -80 °C for 15 min. 
 

SECTION: Lysis and first precipitation
22. Centrifuge the sample at 3500 x g, 45 min, 4 °C. 
 


SECTION: Lysis and first precipitation
23. Discard the supernatant without disturbing the pellet. Use sterile wipes to absorb the liquid on the tube walls, being careful not to disturb the pellet.
SECTION: Lysis and first precipitation
24. To each pellet, add 10 mL G2 buffer, from the QIAGEN kit. Incubate at 50 °C for 30-60 minutes, or until the pellet is dissolved. Swirl the pellet to mix but do not try to pipette or vortex.

SECTION: Column cleanup
25. Equilibrate a QIAGEN Genomic-tip 100/G column with 4 mL of Buffer QBT. 

SECTION: Column cleanup
26. Pour the DNA in G2 buffer through the equilibrated column and allow it to flow through with just gravity.
SECTION: Column cleanup
27. Once all the lysate has passed through, wash the column with 8 mL of Buffer QC.

SECTION: Column cleanup
28. Repeat the wash with another 8 mL of Buffer QC.

SECTION: Column cleanup
29. Place the column over a clean 50-mL Falcon tube, and elute the genomic DNA with 5 mL of Buffer QF, pre-warmed to 55 °C.
SECTION: Column cleanup
30. Allow the eluate to cool down to Room temperature.

SECTION: Column cleanup
31. Add 3.5 mL of isopropanol to the eluted DNA and mix by inverting the tube 10 times.

SECTION: Column cleanup
32. Incubate the tube at -20 °C for at least 3 hours, or 15 min.

SECTION: Final precipitation
33. Centrifuge the tube at 3500 x g, 45 min, 4 °C. 

SECTION: Final precipitation
34. Discard the supernatant without disturbing the pellet.
SECTION: Final precipitation
35. Add 4 mL of ice-cold 70% ethanol to the pelleted DNA and invert the tube 10 times.

SECTION: Final precipitation
36. Centrifuge at 3500 x g, 10 min, 4 °C. 
 


SECTION: Final precipitation
37. Discard the supernatant without disturbing the pellet. Use sterile wipes to dry the tube walls, being careful not to disturb the pellet.
SECTION: Final precipitation
38. Resuspend the DNA in 100 µL of TE buffer and incubate at Room temperature, typically 15 min.

SECTION: Final precipitation
39. Transfer the DNA into a nuclease-free 1.5-mL tube (DNA LoBind tube preferred) using a wide-bore tip, and store at 4 °C. 
 

SECTION: Final precipitation
40. Carry samples forward to BluePippin size selection, if available. This gel separation will retain DNA fragments greater than 15 kb and discard any residual contamination still evident on a Nanodrop trace. For these benefits, expect 50-70% loss of DNA.
SECTION: Lysis and first precipitation
13.1. Invert 10 times every 15 minutes. 
SECTION: Lysis and first precipitation
13.2. At 30 minutes, add another 40 µL of RNase A, inverting 10 times to combine.