TITLE: High molecular weight DNA extraction from plant nuclei isolation optimised for long-read sequencing
AUTHORS: Cedric Mariac

[DESCRIPTION]

High molecular weight DNA was extracted from isolated plant nuclei and used to prepare RAD004 and LSK109 libraries for Nanopore sequencing. Typically, flow cells yielded 3-15 Gb with read lenghts N50 values ranging from 15 to 29 Kb and longest reads > 380 Kb. 


[GUIDELINES]
Pearl millet DNA extracts used for ONT-sequencing

 



ONT sequencing results (Pennisetum glaucum).
 

10 flowcells were loaded : 3 libraries prepared with the RAD4 kit and 7 librairies with the kit LSK109

 





 

 




 

 



























[STEPS]
1. Plant material sampling 
Collect 0.7 g to 1 g of fresh tissue in a 50 ml tube containing liquid nitrogen. 
Grind in a frozen mortar (stored at -80 °C) using a pestle. (Try to avoid mixing tissues with different developmental stages since it might affect DNA yield and quality)

2. Homogenization of fresh tissue 

Transfer powder into an ice-cold tube (50 ml) containing 25 mL ice-cold WB
Filter the homogenate into an ice-cold 50 ml centrifuge bottle through one layer of Scrynel Polyester filter of 25 µm mesh. 

Collect the remaining nuclei by squeezing the homogenate with gloved hands. 

Complete filtrate lysing using a DOUNCE first with loose fitting (A, 10 times) and, then, with a tighter one (B, 10 times)

3. Nuclei extraction 
Pellet the homogenate by centrifugation with a fixed-angle rotor at 2500 x g and 4 °C for 20 min(to be ajusted depending on species)
Discard the supernatant and add 1 mL of ice-cold WB 
Gently resuspend the pellet (you may use a small paint brush) and add up to 25 mL of ice-cold WB. 
Centrifuge 15 min at 2500 x gand 4 °C in a swinging bucket centrifuge. 
Discard supernatant and resuspend pelleted nuclei in 5 mL MATAB lysis buffer supplemented with 17 µLspermine; 17 µL spermidine, and; 20 µL DTT.

4. Nuclei lysis and DNA purification
After gentle homogenization, incubate at 65 °C for 60 min. 
Mix once with extreme precaution.
Add 10 µl of proteinase K (20 mg/ml; eg from Terra PCR Kit, Clonectec) and transfer 55 °C for 105 min. 
Pour gently onto a 15 ml tube.
Add 5 mL of 24 :1 chloroform/isoamylalcool. 
Agitate horizontally at 5 rpm for 10 min.
Centrifuge 20 min at5500 x gand 15 °C. 
Pour the supernatant into a clean 15 ml tube.
Add 10 µL RNase A (Promega, 1mg/ml). 
Mix gently by inversion and incubate for 30 min at RT.
Add 5 mL of 24 :1 chloroform/isoamylalcool and agitate horizontally under chemical hood at 5 rpm for 10 min.
Centrifuge 20 min at 5500 x g and 15°C. Pour the supernatant into a clean 15 ml tube.

(2-3 chloroform extractions in total; if the second extraction is clean enough, the last one can be skipped)


 

5. DNA precipitation

Add 500 µl of 3M NaOAc and 4 ml of isopropanol at room temperature.
Agitate horizontally at 5 rpm until DNA precipitates (medusa).	
Glass hook DNA and transfer into 10 ml 70% EtOH.
Glass hook DNA and transfer into a 2ml-tube with 2 ml of 70% EtOH.	
Centrifuge 10 min at 10000g and discard supernatant. •	
Dry DNA at 45°C for 15 min.
Resuspend in 100µl of sterile H2O.
6. Clean up DNA

Removing contaminants (spermidine, spermine) is mandatory for DNA library preparation using both RAD004 and LSK109 procedures.
Purification was performed using 1X vol/vol AMPure, followed by two 70% ethanol precipation.
DNA was resuspended in 50µl DNAse-free water, and the volume was adjusted to reach a DNA concentration of 300-400 ng/µl.
7. DNA quantification 

DNA was quantified using NanoDrop and Qubit (expected Nano/Qubit ratio < 2).
8. Check HMW using Pulsed-Field Gel Electrophoresis 

Finally,  molecular weight of extracted DNA was assessed by PFGE by loading 20 µl of DNA (1.5 to 5.5 µg of DNA, 5µl of 6x loading buffer) into 1% agarose gel and  using the following parameters for migration:
 
Pulse time : initial = 5, final = 117 
Run time = 20.5 h 
V/cm = 5 
Angle = 120 
Temp = 14° 
mA end of run = 255