TITLE: HMW DNA extraction protocol for ferns
AUTHORS: Geferson Fernando Metz, Rafael Plá Matielo Lemos, Cristiane Barbosa D'Oliveira Matielo, Tiego Ferreira, Filipe Victoria

[DESCRIPTION]
Among the difficulties encountered in a laboratory, simple situations such as efficiently disinfecting fern spores and extracting large amounts of DNA with low tissue input in the protocol are one of the major impediments in carrying out sequencing of these plants in the axenic state. The objective of this work is to present a replicable, scalable and easy-to-execute protocol for work with ferns following the current generation of long-read sequencing. Our method is based on providing a disinfection protocol for spores and sporangia that guarantees growth free of contamination and, after this growth, DNA extraction using a low amount of material in order to obtain a good yield. The results are promising since, in up to 21 days, we obtained germinated plants. After their growth (in average 180 days) we were able to extract DNA in quantity and quality and perform the sequencing, emphasizing that our best N50 is 24 Kb.

[BEFORE_START]
Prepare the SDS Lysis Buffer on the day of the experiment;

    ReagentReagent	Stock ConcentrationFINALPVP 40100%1%Sodium Metabisulfite1%1%NaCl5 M0,5 MTRIS HCL pH 81 M100 mMEDTA pH 80.5 M50 mMDDH2O--SDS*20%1,5%β-MERCAPTOETANOL-2%
When preparing the SDS Lysis Buffer, add SDS at last this will avoiding bubble formation.

Preheat the water bath; Keep the SDS Lysis Buffer at 65°C until the tissue powder is added.

 Washing solution (EtOH 70%), fresh
35 ml Ethanol 100% + 15 ml H2O

Potassium Acetate 5M
Dissolve 4.9 gr KAc in 10 ml ddH2O (4.9 gr KAc + ≈ 7.5 ml H2O).
Adjust the pH with glacial acetic acid.

Prepare TE buffer (10 mM Tris pH 8 and 1 mM EDTA pH 8)
 

[STEPS]
SECTION: Plant material sterilization
3. Recover and store the spores together with sporangia in 1.5 mL microtubes until one-third of the tube was filled and then stored at -20 °C until disinfection.

SECTION: Extraction of high-molecular-weight DNA
9.   
SECTION: Extraction of high-molecular-weight DNA
12. Homogenize by inversion 10 times and incubate at 60 °C for 10 min.

SECTION: Plant material sterilization
4. Add 1 mL  , homogenize by inversion for 20 s  and briefly centrifuge them. 

SECTION: Plant material sterilization
5. Discard the supernatant and wash by inversion with 1 mL of autoclaved purified water,  briefly centrifuge and discard the supernatant.

SECTION: Plant material sterilization
6. Add 1 mL of an    (active chlorine ~2%) at 10 % (v/v) , homogenize by inversion for 20 min , centrifuge at 5.000 rpm 3 min.

SECTION: Plant material sterilization
7. Discard the supernatant and wash by inversion with 1 mL of autoclaved purified water,  centrifuge at 55.000 rpm 3 min. Repeat this step 4 times.

SECTION: Plant material sterilization
8. Add 1 mL autoclaved purified water, homogenize and pipete into a Petri Dish with BCD medium (MgS04.7H2O - 0.1 mM, KH2PO4 - 1.84 mM, KNO3 - 1M, FeSO4.7H2O - 4.5mM).
SECTION: Extraction of high-molecular-weight DNA
10. Weigh between 50 mgand 100 mg of leaf tissue. Grind must be done with a crucible and pestle (previously exposed to   ) until a very fine powder is obtained. 

SECTION: Extraction of high-molecular-weight DNA
11. Pre-heat the Lysis Buffer and aliquot 600 µLfor each sample individually in 1.5 mL microtube. With the aid of a spoon or spatula, transfer the macerate to the microtube.

SECTION: Extraction of high-molecular-weight DNA
13. Add 4 µLof RNase A (100 mg/mL); add 4 µL of proteinase K (> 40 U/mg);

SECTION: Extraction of high-molecular-weight DNA
14. Homogenize by inversion 10 times and incubate at 60 °C for 20 min gently homogenizing by inversion every 5 min.

SECTION: Extraction of high-molecular-weight DNA
15. Add 600 µLof   and homogenize by inversion for at least 3 min until an off-white emulsion forms.

SECTION: Extraction of high-molecular-weight DNA
16. Centrifuge at 14.000 rpmfor 5 minat room temperature.

SECTION: Extraction of high-molecular-weight DNA
17. Carefully aspirate the upper phase of the tube and transfer to a new 1.5 mL microtube.
SECTION: Extraction of high-molecular-weight DNA
18. Add 400 µLof   -20 °C and incubate it for at least 60 min at -20 °C. (Can be stored overnight)

SECTION: Extraction of high-molecular-weight DNA
19. Centrifuge at 14.000 rpmfor 5 min at room temperature and discard the supernatant.

SECTION: Extraction of high-molecular-weight DNA
20. Add 50 µLof  . Add 5 µLof  .

SECTION: Extraction of high-molecular-weight DNA
21. Add 120 µLof iced   (-20 °C) and mix well by flicking the tube.

SECTION: Extraction of high-molecular-weight DNA
22. Store tubes at -20 °C for at least 20 min.

SECTION: Extraction of high-molecular-weight DNA
23. Centrifuge at 14.000 rpm for 15 min at room temperature and carefully discard the supernatant.

SECTION: Extraction of high-molecular-weight DNA
24. Add 1 mL of freshly prepared  , mix gently by inversion, centrifuge briefly and carefully discard the supernatant.

SECTION: Extraction of high-molecular-weight DNA
25. 50 µL Dry the pellet for approximately 10 minat Room Temperature on the bench with the tube upside down.

SECTION: Extraction of high-molecular-weight DNA
26. Elute in 50 µL volume in  . (Elution volume can be adjusted for your needs)

SECTION: Quality Assessment
27. Quantify the DNA on a Qubit® fluorometer (dsDNA high sensitivity assay). DNA yield can be 500 - 1500 ng.
SECTION: Quality Assessment
28. Check DNA integrity though electrophoresis in a 1'% Agarose Gel.
SECTION: DNA Size Selection
29. Remove short DNA fragments with Circulomics® Short-Read Eliminator Kit.
SECTION: Plant material sterilization
1.   

SECTION: Plant material sterilization
2. Sample and store the leaf tissue (fronds) in paper envelopes for three days to induce dehiscence.