Five grams of flash frozen leaf tissue was ground in liquid nitrogen and extracted with 20 mL CTAB/Carlson lysis buffer (100 mM Tris-HCl, 2% CTAB, 1.4 M NaCl, 20 mM EDTA, pH 8.0) containing 20 μg/mL proteinase K for 20 min at 55 °C. The DNA was purified by addition of 0.5× volume chloroform, which was mixed by inversion and centrifuged for 30 min at 3000 RCF, and followed by a 1× volume 1:1 phenol: 24:1 chloroform:isoamyl alcohol extraction. The DNA was further purified by ethanol precipitation (1/10 volume 3 M sodium acetate pH 5.3, 2.5 volumes 100% ethanol) for 30 min on ice. The resulting pellet was washed with freshly prepared ice-cold 70% ethanol, dried, and resuspended in 350 μL 1× TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) with 5 μL RNase A (Qiagen, Hilden) at 37 °C for 30 min, followed by incubation at 4 °C overnight. The RNase A was removed by double extraction with 24:1 chloroform:isoamyl alcohol, centrifuging at 22,600×g for 20 min at 4 °C each time. An ethanol precipitation was performed as before for 3 h at 4 °C. The pellet was washed as before and resuspended overnight in 350 μL 1× TE.
Genomic DNA sample was further purified for ONT sequencing with the Zymo Genomic DNA Clean and Concentrator-10 column (Zymo Research, Irvine, CA).