TITLE: High molecular weight plant DNA extraction for PacBio HiFi sequencing
AUTHORS: Kanae Nishii, Michael Möller, Robert G. Foster, Laura L. Forrest, Nathan Kelso, Sadie Barber, Caroline Howard, Michelle L. Hart

[DESCRIPTION]
High molecular weight and high quality DNA is mandatory for successful long-read sequencing. In addition, PacBio HiFi SMRTBell library construction requires omission of traditional DNA extraction chemicals such as guanidinium, chloroform and others. We developed a DNA extraction protocol working well for the recalcitrant plant Streptocarpus, and the extracted DNA was successfully used for PacBio HiFi sequencing.

[BEFORE_START]
Prepare and check plastic and chemicals listed under section “Materials”.
Ready-to-use nuclei isolation (NI) buffer and sorbitol buffer containing β-mercaptoethanol, spermidine, spermine, and PVP40 should be prepared on the day of DNA extraction.
Leave NI buffer and Triton X-100/NIB on ice for precooling.
Affix 100 µm pore size nylon mesh on top of a 500 ml glass beaker with string or tape. Mesh can be replaced with Corning cell strainers (100 µm pore size) and 50 ml Falcon tubes.
Depending on plant species and if possible, it is recommended to cultivate > 100 g plant material to allow protocol optimization. 


[GUIDELINES]
This is a DNA extraction protocol designed for Streptocarpus PacBio HiFi sequencing on the Sequel II system.

[STEPS]
SECTION: Before starting DNA extraction
1. Prepare 400 ml NI buffer in a glass beaker and leave on ice.
on ice 

SECTION: Before starting DNA extraction
2. Prepare 100 ml sorbitol buffer in two 50 ml Falcon tubes.
on ice 

SECTION: Before starting DNA extraction
3. Affix nylon mesh on top of one empty 500 ml glass beaker with string or tape (or place Corning cell strainer on 50 ml Falcon tube).
SECTION: Before starting DNA extraction
4. Arrange liquid nitrogen and mortar and pestles on fume bench.
SECTION: Tissue grinding
5. Grind fresh leaf tissue in liquid nitrogen and mortar and pestle 3 times to a fine powder and add ground tissue sample to NI buffer prepared at step 1. Grind 1-2 grams leaf tissue at a time and in total approx. 30 grams in this protocol.
on ice 

SECTION: Tissue grinding
6. Filter sample-NI buffer mix through 100 µm pore size nylon mesh/beaker prepared at step 3. Keep all solutions on ice during filtering.
on ice 

SECTION: Tissue grinding
7. Divide filtrate equally to ten 50 ml Falcon tubes kept on ice.
on ice 

SECTION: Tissue grinding
8. Add 1/20th volume of 10% Triton X-100/NIB to tubes prepared at step 7. Gently mix by inverting tubes.
on ice 

SECTION: Tissue grinding
9. Centrifuge tubes at 2,000 × g for 10 minutes at 4 °C.
2000 x g, 10 min, 4 °C 

SECTION: Tissue grinding
10. Discard supernatant gently by decantation, without disturbing or losing pellet.
SECTION: Tissue grinding
11. Add 10 ml sorbitol buffer to each tube and mix gently.
SECTION: Tissue grinding
12. Centrifuge tubes at 3,000 × g for 10 minutes at 4 °C.
3000 x g, 10 min, 4 °C 

SECTION: Tissue grinding
13. Discard supernatant by decantation (Optionally, repeat sorbitol buffer wash until supernatant is clear).
SECTION: CTAB lysis
15. Add 3 ml CTAB lysis buffer directly to frozen pellet, a pinch of PVPP, and 12 µl RNase A to each tube and mix well by gently pipetting with wide-bore tips. Incubate at 58 °C for 20 minutes.
58 °C 

SECTION: CTAB lysis
16. Add 60 µl proteinase K to each tube. Incubate for more than 3 hours, but less than 5 hours, at 58 °C. Occasionally shake tubes gently.
58 °C 

SECTION: CTAB lysis
17. Centrifuge at 4,400 × g for 10 minutes at room temperature. Collect clear lysate to new 50 ml Falcon tube avoiding any cell debris.
4400 x g, 10 min 

SECTION: CTAB lysis
18. To maximize lysate recovery, move remained debris/lysate to 2 ml tubes and centrifuge at 11,000 rpm for 5 minutes. Move clear lysate to same tube at step 17. In total approx. 30 ml lysate can be obtained.
11000 rpm, 5 min 

SECTION: CTAB lysis
19. Adjust lysate with 0.25 N HCl to between pH 7.0 - 7.5. Check pH with pH indicator strips. Add 1 ml or less 0.25N HCl at a time and check with pH paper each time.


SECTION: CTAB lysis
20. Divide lysate to two 50 ml Falcon tubes. Add equal volume of Milli-Q water. 

NOTE: Do not centrifuge tubes once water is added. Low salt condition tends to promote formation of a CTAB-DNA solidified complex.
SECTION: Qiagen Genomic-tip 100/G DNA extraction
21. Proceed with Qiagen Genomic-tip 100/G following the manufacturer`s protocol. Set up six empty 50 ml Falcon tubes, labelled “QBT”, “Sample”, “QC1”, “QC2”, “QC3”, “Final DNA”. Set up three sets of each.
SECTION: Qiagen Genomic-tip 100/G DNA extraction
22. Set water bath to 50 °C and prewarm buffer QF.
50 °C 

SECTION: Qiagen Genomic-tip 100/G DNA extraction
23. Place Genomic-tip 100/G column on 50 ml Falcon tube labelled “QBT”. Equilibrate Genomic-tip 100/G column with 4 ml buffer QBT. Allow buffer to flow through column completely by gravity.
SECTION: Qiagen Genomic-tip 100/G DNA extraction
24. Move Genomic-tip 100/G to next Falcon tube labelled “Sample”. Load one third of lysate (approx. 20 ml) obtained at step 20 to one Genomic-tip 100/G. Allow lysate to flow through column completely by gravity.
SECTION: Qiagen Genomic-tip 100/G DNA extraction
25. Move Genomic-tip 100/G to tube labelled “QC1”. Load 7.5 ml buffer QC onto column. Allow buffer to flow through column completely by gravity.
SECTION: Qiagen Genomic-tip 100/G DNA extraction
26. Repeat buffer QC step two more times, on tubes labelled “QC2” and “QC3”. In total, DNA in Genomic-tip 100/G column should be washed three times with buffer QC.
SECTION: Qiagen Genomic-tip 100/G DNA extraction
27. For final DNA elution, apply 5 ml QF buffer prewarmed to 50 °C to each Genomic-tip 100/G column.
50 °C 

SECTION: Qiagen Genomic-tip 100/G DNA extraction
28. Divide eluted DNA in 1 ml aliquots to 2 ml tubes. Add 0.7 volume (0.7 ml) ice-cold isopropanol. Gently invert and mix and leave tubes at -20 °C overnight. 
-20 °C 
[SAFE STOP POINT for at least a few days]
SECTION: Qiagen Genomic-tip 100/G DNA extraction
29. Centrifuge tubes at 11,000 rpm for 10 minutes.
11000 rpm, 10 min 

SECTION: Qiagen Genomic-tip 100/G DNA extraction
30. Discard supernatant and add 1 ml 70% ethanol.
SECTION: Qiagen Genomic-tip 100/G DNA extraction
31. Centrifuge at 11,000 rpm for 10 minutes.
11000 rpm, 10 min 

SECTION: Qiagen Genomic-tip 100/G DNA extraction
32. Discard supernatant and air-dry pellet by inverting tubes on clean tissue. Warming tubes at 37 °C for 10 minutes speeds up evaporation of ethanol, but do not over-dry.
SECTION: Qiagen Genomic-tip 100/G DNA extraction
33. Add 15-20 µl Low (0.1 ×) TE buffer.
SECTION: Qiagen Genomic-tip 100/G DNA extraction
34. Incubate tubes at 50 °C, and 300 rpm for 1 hour. Collect eluted DNA to 1.5 ml LoBind Eppendorf tube.
50 °C 

SECTION: Qiagen Genomic-tip 100/G DNA extraction
35. Add 15-20 µl Low (0.1 ×) TE buffer for 2ndelution.
SECTION: Qiagen Genomic-tip 100/G DNA extraction
36. Incubate tubes at 50 °C, and 300 rpm for 1 hour. Collect DNA elute to 1.5 ml LoBind Eppendorf tube. Keeping 1stand 2nd DNA elution in separate tubes is recommended.
50 °C 

SECTION: DNA quality control (QC)
37. Proceed to DNA quality control. DNA quantification with Qubit and Nanodrop. For DNA quality, obtain A260/A280 and A260/A230 values with Nanodrop. Examine DNA fragment size distribution with TapeStation or FemtoPulse.
SECTION: Tissue grinding
14. To remove sorbitol buffer completely, invert tubes on dry tissue briefly but take care not to lose pellet. Pellet of nuclei and small cells remaining in tubes can now be frozen in liquid nitrogen and stored at -80 °C. 
-80 °C 

[SAFE STOP POINT for at least a few days]