TITLE: Plant DNA extraction and preparation for ONT sequencing
AUTHORS: Boas Pucker

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Plant DNA extraction and preparation for ONT sequencing</span></div><div class = "text-block">This CTAB-based protocol is suitable for extraction of genomic DNA from a wide range of plant species. The DNA quality is sufficient for ONT sequencing after SRE enrichment of long fragments and removal of short fragments. Quality control steps are described as part of this protocol.</div><div class = "text-block">Successful application for the genome sequencing of the following plant species:</div><div class = "text-block"><span style = "font-style:italic;">Arabidopsis </span><span>species, </span><span style = "font-style:italic;">Beta vulgaris</span><span> (sugar beet), </span><span style = "font-style:italic;">Brassica napus</span><span> (rapeseed/canola), </span><span style = "font-style:italic;">Dioscorea dumetorum</span><span> (yams), </span><span style = "font-style:italic;">Helichrysum umbraculigerum, </span><span>mosses, </span><span style = "font-style:italic;">Vitis vinifera</span><span> (grapevine)</span></div><div class = "text-block"><span style = "font-weight:bold;">References:</span></div><div class = "text-block">Siadjeu C.*, Pucker B.*, Viehoever P., Albach D. and Weisshaar B. (2020).   High contiguity de novo genome sequence assembly of Trifoliate yam (Dioscorea dumetorum) using long read sequencing. Genes. doi:10.3390/genes11030274.</div><div class = "text-block">Pucker B, Rückert C, Stracke R, Viehöver P, Kalinowski J,  Weisshaar B.  Twenty-Five Years of Propagation in Suspension Cell Culture Results in  Substantial Alterations of the Arabidopsis Thaliana Genome.  Genes. 2019. doi:10.3390/genes10090671.</div><div class = "text-block">Photo provided by Hanna Schilbert (@HSchilbert).</div></div>

[STEPS]
?. [CTAB-based DNA extraction]
Preheat 5 ml CTAB1 + 300 µL ß-ME in 50 ml tube to 75 C(The amount of ß-ME is working, but was not optimized)
?. [CTAB-based DNA extraction]
Homogenize fresh material with morta and pestle in liquid N2(It is important to use fresh material which was not frozen previously. Less than 1g of young leaves is usually sufficient. Keeping plants in the dark for a few days prior to DNA extraction might reduce the amount of starch and the number of plastids.)
?. [CTAB-based DNA extraction]
Add homogenized material (powder) to the preheated CTAB1 buffer and resuspend carefully(It is crucial to completely resuspend the powder. Avoid transfer of ice.)
?. [CTAB-based DNA extraction]
Incubation for at least 30 minutes (up to 2 hours possible). Invert tubes frequently to carefully mix the solution.
?. [CTAB-based DNA extraction]
Re-cool to room temperature
?. [CTAB-based DNA extraction]
Add 5 ml dichloromethane and mix gently by inverting the tube.
?. [CTAB-based DNA extraction]
Centrifugation for 30 minutes at >10,000 x g at room temperature
?. [CTAB-based DNA extraction]
Transfer upper phase carefully to new 50 ml tube.(Do not disturb interphase. Upper phase should be colorless.)
?. [CTAB-based DNA extraction]
Add 10 ml CTAB2 and mix gently by inverting the tube.
?. [CTAB-based DNA extraction]
Centrifugation for 30 minutes at >10,000 x g at room temperature
?. [CTAB-based DNA extraction]
Discard supernatant.
?. [CTAB-based DNA extraction]
Add 2 ml 1M NaCl and re-dissolve pellet.
?. [CTAB-based DNA extraction]
Add 2 ml Isopropanol and mix gently.
?. [CTAB-based DNA extraction]
Centrifugation for 30 minutes at >10,000 x g at room temperature
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Discard supernatant.
?. [CTAB-based DNA extraction]
Wash with 2ml of 70% ethanol.
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?. [CTAB-based DNA extraction]
Wash with 2 ml 70% ethanol.
?. [CTAB-based DNA extraction]
Centrifugation for 10 minutes at >10,000 x g at room temperature
?. [CTAB-based DNA extraction]
Discard supernatant.
?. [CTAB-based DNA extraction]
Brief centrifugation to collect droplets.
?. [CTAB-based DNA extraction]
Remove droplets.
?. [CTAB-based DNA extraction]
Dissolve sediment in 100 µl CTAB-TE via incubation over night at room temperature.(RNase is active during this time)
?. [Quality control]
NanoDrop measurement with 1 µl of DNA solution(expecation: several µg/µl)
?. [Quality control]
Agarose gel: 1 µl DNA + 3 µl loading buffer + 3 µl water(1kb DNA ladder from NEB as size control; any large DNA fragment will do the job)
?. [Quality control]
Qubit measurement with 1 µl DNA
?. [SRE]
Removal of short fragments following the SRE (Circulomics) handbook:https://www.n-genetics.com/products/1317/1023/17106.pdf
?. [ONT sequencing]
LSK109 works best for plant genome sequencing (in my hands).Two libraries may be prepared for the cost of one by starting with 2 µg of DNA (as recommended for R10 flow cells) even when using R9 flow cells. This comes with the risk that not all pores will be occupied depending on the library quality. The volumen of the final step (Elution Buffer) is increased to 25 µl. Half of the library (12 µl) is used for sequencing right away and the rest of the library can be stored at 4 C in a fridge until needed after a nuclease flush. There might be a small decrease in average read length due to storage of the library.
?. [ONT sequencing]
Nuclease flush might be necessary after some time (e.g. 12-24 hours) depending on the DNA quality. Incubation with DNaseI on the flow cell for 2 hours works well in my hands.