Fungal DNA extraction for Nanopore sequencing

The frozen mycelium samples are ground to fine powders using a mortar and pestle in liquid nitrogen to avoid raising the temperature of the samples for high molecular weight DNA extraction.

Add 20mL of pre-warmed (60°C) lysis buffer with 800µL ProteinaseK (FUJIFILM Wako Pure Chemical Co., Ltd. Japan) and 50µL RNase A (Nippon Gene Material Co., Ltd. Japan) in a glass beaker.

20mL lysis buffer [2% CTAB, 100mM TrisHCl, 20mM EDTA, 1.4M NaCl, 1% PVP)]

800µL ProteinaseK

50µL RNase A

60°C

The mixture was incubated at 55 °C in a water bath with shaking for 5 min.

55°C

0h 5m 0s.

Add 1 vol phenol/chloroform/isoamylic alcohol (PCI) 25:24:1 and mix by inversion.

Centrifuge 12000 rpm (13000 g) with soft/slow break function for 20 min at RT.

12000rpm,0h 0m 0s 13000x g,0h 0m 0s

0h 20m 0s

Room temperature

Remove aqueous layer to new 50 mL tube.

Repeat step 4

Add 1 vol chloroform and mix by inversion.

Centrifuge 14000 rpm (17800 g) with soft/slow break function for 10 min at RT.

14000rpm,0h 0m 0s 17800x g,0h 0m 0s

0h 10m 0s

Room temperature

Remove aqueous layer to new 50 mL tube.

Repeat step 5

Add 1 vol isopropanol and mix gently by inversion.

Centrifuge 14000 rpm for 5 min at 4 °C. Remove and discard the supernatant.

14000rpm,0h 0m 0s 17800x g,0h 0m 0s

0h 5m 0s

4°C

Rinse pellet with 1mL 75% ethanol at RT.

1mL 75% ethanol

All aqueous and pellet to new 2 mL tube.

Spin down and remove supernatant.

Air dry DNA pellet.

Resuspend DNA in sterile 50uL 10mM Tris-HCl for 1 hr~ at RT in dark.

50µL Tris-HCl

Room temperature

1h 0m 0s~1h 0m 0s

Check the quality and concentration of DNA using spectrophotometrically with NanoDrop (Thermo Fisher Scientific, USA) and in a Qubit 2.0 fluorometer (Thermo Fisher Scientific).

Check the DNA degradation using Genomic DNA ScreenTape assay with 2200 TapeStation system (Agilent Technologies, Germany).

Store at 4 °C until library preparation. (at -20 °C for long-term storage)