TITLE: Extraction and selection of high-molecular-weight  DNA for long-read sequencing from Chlamydomonas reinhardtii 
AUTHORS: Frédéric Chaux-Jukic, Nicolas Agier, Stephan Eberhard, Zhou Xu

[DESCRIPTION]
Recent advances in long-read sequencing technologies have enabled the complete assembly of eukaryotic genomes from telomere to telomere by allowing repeated regions to be fully sequenced and assembled, thus filling the gaps left by previous short-read sequencing methods. Furthermore, long-read sequencing can also help characterizing structural variants, with applications in the fields of genome evolution or cancer genomics. For many organisms, the main bottleneck is to develop robust methods to obtain high-molecular-weight (HMW) DNA for whole genome sequencing purposes. We developed an optimized protocol to extract DNA suitable for long-read sequencing from the unicellular green alga Chlamydomonas reinhardtii, based on CTAB/phenol extraction followed by a size selection step for long DNA molecules. We provide validation results for the extraction protocol, as well as statistics obtained with Oxford Nanopore Technologies sequencing.

[BEFORE_START]
On the day of extraction: 
- Set water bath to 65°C (large enough for one 50-ml tube per sample)
- Chill isopropanol to -20°C (around 2.5 ml per sample)


[GUIDELINES]
- This protocol aims at extracting high-molecular-weight DNA molecules with maximal removal of contaminants. Both parameters are indeed crucial to take full advantage of long-read sequencing.
- This protocol was developed for the microalga Chlamydomonas reinhardtii, but might be adapted to other algae and land plants.
- This protocol is relatively simple and short, however, for basic molecular biology (PCR, cloning of small fragments, etc.), simpler protocols may be preferred (e.g. www.chlamycollection.org/methods/).
- The extraction steps rely mostly on home-made solutions. Purification and size selection uses commercial reagents, the composition of which is unknown to us.

[STEPS]
SECTION: Harvesting and storage of cells
1. Grow Chlamydomonas reinhardtii cells in 100 mL TAP medium under low light (~5 µmol photon.m-2.s-1) with constant shaking at  , or in other conditions as appropriate for the specific experiment.

SECTION: Harvesting and storage of cells
2. Harvest the cells at the end of exponential growth phase (~107 cells.mL-1) in 50 mL tubes by centrifugation 4000 x g, 5 min. Discard supernatant.

SECTION: Harvesting and storage of cells
3. Store cell pellets at -20 °C (optional).

SECTION: DNA extraction
4. Thaw cell pellets at Room temperature  (not applicable if the pellets were not frozen) and put on ice.

SECTION: DNA extraction
5. Centrifuge at 4000 x g, 5 min, 4 °C and discard any liquid left.

SECTION: DNA extraction
6. Resuspend by gentle pipetting in 3 mL of CTAB solution preheated at 65 °C.
 

SECTION: DNA extraction
7. Add 5 µL of proteinase K (stock solution: 20 mg.mL-1) and 5 µL  of RNase A (stock solution: 100 mg.mL-1), mix by pipetting and incubate at 65 °C for 20 min in a water bath. 


SECTION: DNA extraction
8. Centrifuge at 4000 x g, 5 min and distribute 3 x 1 mL of each supernatant into 2 mL tubes. Discard pellet.

 



SECTION: DNA extraction
9.  Add 1 mL of phenol:chloroform:isoamyl alcool (25:24:1) to each tube, mix gently by inverting 10 times and centrifuge at 20000 x g, 5 min, 4 °C. Transfer the aqueous (upper) phase to new 2 mL tubes.  
 
 
 



SECTION: DNA extraction
10. Add 1 µL of proteinase K and 1 µL  of RNase A, mix gently by inverting 3 times and incubate at 50 °C for 20 min.

 

SECTION: DNA extraction
11. Add 1 mL of chloroform:isoamyl alcohol (24:1) to each tube, mix gently by inverting 3 times and centrifuge at 20000 x g, 5 min, 4 °C. Transfer the upper phase to new 2 mL tubes.

SECTION: DNA extraction
12. Add 700 µL of isopropanol to each tube and mix gently by inverting 10-12 times. A visible DNA precipitates should form.

 

SECTION: DNA extraction
13. Centrifuge at 20000 x g, 5 min, 4 °C, discard the supernatant carefully, add 500 µL of ice-cold 70% ethanol without disturbing the pellet and centrifuge at 20000 x g, 5 min, 4 °C.

SECTION: DNA extraction
14. Discard the ethanol carefully by pipetting and eliminate the remaining traces using a SpeedVac for 5 min at Room temperature  or leave tubes open for 30 min  at Room temperature .

SECTION: DNA extraction
15. Resuspend the pellet in 30 µL of ultrapure water overnight at Room temperature.



SECTION: Purification and selection of high-molecular-weight DNA
16. Pool the samples corresponding to the same initial culture in one 1.5ml Lo-Bind tube, add an equal volume (e.g. 3 x 30 µL) of AMPure XP beads, mix gently by hand for 5 min.

 

SECTION: Purification and selection of high-molecular-weight DNA
17. Place the tubes on a magnetic rack and let the beads aggregate on the side of the tube for 5 min. Discard the supernatant. Remove the tubes from the magnetic rack, resuspend the beads in 60 µL of ultrapure water for at least 15 min at Room temperature.

SECTION: Purification and selection of high-molecular-weight DNA
18. Place the tubes on a magnetic rack and let the beads aggregate on the side of the tube for 5 min. Transfer the supernatant to new 1.5 mL Lo-Bind tubes and discard the beads.
SECTION: Purification and selection of high-molecular-weight DNA
19. Measure DNA concentration and purity using a Nanodrop and/or a Qubit device.
SECTION: Purification and selection of high-molecular-weight DNA
20. Adjust each sample to ~150 ng.µL-1 in 60 µL of ultra-pure water, gently mix with an equal volume of Short Read Eliminator (SRE) buffer (SRE kit, Circulomics) and centrifuge at 10000 x g, 30 min, 4 °C. Pipet out the supernatant very carefully as the pellet is colorless and can easily be lost. 

 

SECTION: Purification and selection of high-molecular-weight DNA
21. Gently add 200 µL of freshly prepared 70% ethanol to the pellet. Wash by centrifuging at 10000 x g, 2 min, 4 °C and discarding the ethanol.
SECTION: Purification and selection of high-molecular-weight DNA
22. Repeat Step 21.
SECTION: Purification and selection of high-molecular-weight DNA
23.  Air dry the pellet for 10 min at Room temperature and resuspend in 50 µL of pre-warmed EB buffer (SRE kit, Circulomics) by incubating at 50 °C for 20 min.
SECTION: Purification and selection of high-molecular-weight DNA
24. Proceed to long-read sequencing or store at 4 °C for later use (at least several weeks). Do not freeze to avoid DNA shearing.



SECTION: Quality check
25. DNA purity can be assessed by Qubit or Nanodrop.
SECTION: Quality check
26. Absence of small fragments can be assessed by electrophoresis on agarose gel and size distribution can be assessed by pulsed-field gel electrophoresis (PFGE).