Frozen tissue was ground to a fine powder in liquid nitrogen and added to 30 mls of ice-cold nuclei isolation buffer (WPB buffer, Loureio et al., 2007) and mixed by inversion. The mixture was then homogenized using a Tekmar Homigenizer set to 80% power for 2 minutes and then the mixture was passed through a 40 um mesh filter and then the flowthrough was centrifuged at 3,200g for 20 minutes at 4C. The supernatant was removed and the pellet gently resuspended in 30 ml WPB and centrifuged at 3,200g, 20 min, 4C. The supernatant was removed and 30 ml of HMW DNA extraction buffer was added (Schalamum and Schwessinger, 2017, Baptiste et al., 2016) and the pellet resuspended. RNase A was added (10 ul of 100mg/ml stock) and the solution incubated for 20 min at 37C. Protease K was then added (30 ul, 20 mg/ml stock) and the solution incubated for an additional 30 minutes at 37C. Proteins were precipitated through the addition of 1/3 vol 5M KOAc- and the solution incubated on ice for 10 minutes followed by centrifugation at 3,200g for 15 minutes at 4C. The supernatant was transferred to a new tube and 0.7 x volume of isopropanol was added to precipitate the DNA at 4C overnight. The solution was centrifuged at 3,200 g for 20 minutes and the supernatant removed. The pellet was washed with twice with 80% EtOH to remove salts and 1 time with 95% EtOH to add in drying. The pellet was dissolved in 500 ul low TE. To remove polysaccharides a salt cleanup was performed (Fang et al., 1992). Briefly, 500 ul of 5M NaCl was added to the DNA solution and gently mixed. The solution was centrifuged at 8,000 g for 20 minutes and the supernatant was transferred to a new tube. DNA was precipitated with isopropanol (0.7x vol) and washed twice with 80% EtOH and once with 95% EtOH as described above. The final pellet was dissolved in 50 ul of low TE and used for QC and library preparation or a circulomics SRE size selection (Short Read Eliminator Kit (circulomics.com)) can be performed prior to library preparation to remove any fragments below 10Kb.
Citations
Baptiste Mayjonade, Jérome Gouzy, Cécile Donnadieu, Nicolas Pouilly, William Marande, Caroline Callot, Nicolas Langlade and Stéphane Munos, High molecular weight gDNA extraction, Bio Techniques, Vol. 61, No. 4, October 2016, pp. 203-205
Fang G, Hammar S, Grumet R. A quick and inexpensive method for removing polysaccharides from plant genomic DNA. Biotechniques. 1992 Jul;13(1):52-4, 56. PMID: 1503775.
Leblanc O, Zekraoui L, Mariac C. High molecular weight DNA extraction from plant nuclei isolation optimised for long-read sequencing. Protocols.io. 2020 https://www.protocols.io/view/high-molecular-weight-dna-extraction-from-plant-nu-83shyne
Loureio J, Rodrifuez E, Dolezel J, Santos C. Two new Nuclear Isolation Buffers for Plant DNA Flow Cytometry: A Test with 37 Species. 2007. Annals of Botany 100:875-888
Schalamum M, Schwessinger. High molecular weight gDNA extraction after Mayjonade et al. optimized for eucalyptus for nanopore sequencing Vsersion 9. Protocols.io. 2017 https://www.protocols.io/view/high-molecular-weight-gdna-extraction-after-mayjon-khkct4w/guidelines