TITLE: Wheat leaf high-molecular weight (HMW) DNA extraction
AUTHORS: Maximillian RW Jones, Nikolai Adamski, Cristobal Uauy

[DESCRIPTION]
This protocol is for the extraction of high-molecular weight DNA from wheat leaf tissue. The product is suitable for long-read sequencing such as PacBio. The protocol may also be effective for leaves of other cereal species. For example, we have had success using it with tef (Eragrostis tef).

The protocol consists of four phases; 1) buffer preparation, 2) harvesting of leaf tissue, 3) crude nuclei extraction, and 4) HMW extraction. The last of these uses the PacBio Nanobind® plant nuclei kit.

Using this protocol we have produced good quality DNA extractions with the following features:
Fragment size distribution peak >50 kb (Agilent Tapestation, Genomic DNA ScreenTape)
Concentration between 350 and 600 ng / µl (Invitrogen Qubit 3 fluorometer)
260/280 ratio 1.75-1.90 and 260/230 ratio 1.75-1.90 (Thermo Scientific Nanodrop)

This protocol was originally adapted from Dvorak et al., 1988, but has been modified over many years.

[BEFORE_START]
Please see the Materials section for instructions on buffer preparation, storage conditions, and longevity.

We highly recommend you read through the whole protocol before attempting it as certain steps are complex and time-critical.

[STEPS]
SECTION: Buffer preparation
30. Prepare 10x HB

SECTION: Buffer preparation
0.30. 
SECTION: Buffer preparation
0.30. 
SECTION: Buffer preparation
0.30. 
SECTION: Tissue collection
1. Sanitise scissors and/or other tools, plus gloved hands, with 70% ethanol. Collect 2-5 g leaf blade tissue into a 50 mL Falcon tube. Do not use leaf sheath tissue. Avoid using diseased, bleached, or senescing sections. According to PacBio, younger leaves generally produces higher DNA quality and quantity. Avoid bruising tissue as much as possible. Using forceps may help with this. Re-sanitise tools and hands between samples.
SECTION: HMW DNA extraction
20. For the extraction of HMW DNA from the nuclei prep, we utilised PacBio's Nanobind® plant nuclei kit and protocol with one minor modification.

Overview:
https://www.pacb.com/wp-content/uploads/Guide-overview-Nanobind-plant-nuclei-kit.pdf
Protocol:
https://www.pacb.com/wp-content/uploads/Procedure-checklist-Extracting-HMW-DNA-from-plant-nuclei-using-Nanobind-kits.pdf

Modifications:
Use 2 mL rather than 1.5 mL Protein LoBind tubes. The more rounded bottom prevents the Nanobind disks becoming lodged.
SECTION: Crude nuclei extraction
19. Either proceed directly to Step 24, or snap freeze the nuclei pellets in liquid nitrogen and store at -70 to -80 °C to continue the protocol at a later date. We have only tested using frozen nuclei pellets up to 1 week old, but they may be viable for longer.
SECTION: Crude nuclei extraction
10. Spin samples for 20 minutes at 1500 x g at 4 °C. If necessary for balance, adjust volumes with iced 1xHB/β-ME and/or use H2O-filled balance bombs.
SECTION: Crude nuclei extraction
9. Repeat until all samples done. Samples should be incubated on ice for at least 10 minutes before moving onto Step 14. 
SECTION: Crude nuclei extraction
8. Filter the ground sample through the filtration sandwich into the centrifuge bomb. While this is dripping through, place blender under a fast-flowing tap, being sure to also rinse the lid. After filtration is complete, squeeze out remaining sample liquid from cloth into the waste beaker and discard cloth into a chemical waste bin. Thoroughly rinse the funnel and your gloved hands. Wipe scissor blades with a paper towel soaked in 70% ethanol. Cap the centrifuge bomb, gently shake, and place back on ice.
SECTION: Crude nuclei extraction
5. Place funnel into one of the 250 mL centrifuge bombs, still on ice. Insert the Mira/cheese-cloth sandwich and push down with the glass rod or gloved finger.
SECTION: Crude nuclei extraction
7. Cut plant tissue into small pieces directly into the blender. Start blending on a low setting until the plant tissue is incorporated into suspension. Then blend on high for one minute.
SECTION: Crude nuclei extraction
6. Measure out 100 mL 1xHB/β-ME into the blender (for 2-5 g tissue - if using more tissue increase to 150 mL)

SECTION: Crude nuclei extraction
4. Working in the fume hood, add 640 µL β-mercaptoethanol to 160 mL of 1xHBS for each sample and mix well. This mixture is referred to as 1xHB/β-ME for the remainder of this protocol.
SECTION: Crude nuclei extraction
3. Set up centrifuge with correct rotor and swing bucket attachments and begin cooling
to 4 °C.  Place 5 mL of H+20 buffer into a 250 mL centrifuge bottle per sample. Label accordingly. Place 
on ice in an ordered manner.
SECTION: Crude nuclei extraction
2. Set up fume hood with the following:
Blender (~1 L volume, preferably glass-walled as some plastics may react with β-mercaptoethanol)
Tissue samples on ice
1xHB on ice (100 mL per sample)
H+20 buffer on ice (5 mL per sample)
β-mercaptoethanol (400 µL per sample)
250 mL conical centrifuge bombs on ice (1 per sample) (e.g. Corning CLS430776) (do not use
flat-bottomed centrifuge bottles)
Mira/cheese-cloth filtration sandwiches (1 per sample)
Large plastic funnel
100 mL graduated cylinder
Glass rod
Scissors
Timer
70% ethanol for cleaning
Blue roll
1 L waste beaker

If using frozen tissue, finish preparing the fume hood before removing samples from freezer.
SECTION: Tissue collection
1.2. If conducting nuclei extraction at a later date, snap freeze the Falcon tube in liquid nitrogen then store at -70 to -80 °C.
SECTION: Tissue collection
1.1. If conducting nuclei extraction immediately, bury Falcon tube up to cap in ice.
10. 
SECTION: Crude nuclei extraction
11. For each sample bottle, carefully pour off the supernatant into the waste
bucket, then invert onto the stacked paper towels to remove excess liquid.
SECTION: Crude nuclei extraction
10.1. While samples are spinning, tidy the fume hood and re-prepare it with the following:
1xHB/β-ME on ice
Additional ice buckets with enough space for all samples
Five-layer stacks of paper towels for blotting sample bottles
Small paint brushes soaking in 1xHB
P1000 pipette
1000 µL tips with cut ends
1 L waste beaker
SECTION: Crude nuclei extraction
12. Add 1 mL of ice-cold 1xHB/β-ME to each centrifuge bottle. Use a clean paint brush (soaked in 1xHB) to resuspend the pellet. Next, mix using a P1000 pipette using a cut-tip until solution is homogeneous. Transfer sample to a 50 mL pre-labelled Oakridge tube.
SECTION: Crude nuclei extraction
13. Bring the volume of each sample to approximately 15 mL using ice-cold 1xHB/β-ME. Spin for 10 min at 1100 x g at 4°C.
SECTION: Crude nuclei extraction
14. Repeat Steps 15 to 17 twice more. The supernatant should be transparent after the
last centrifugation. The same paint brush can be re-used for the same sample.
SECTION: Crude nuclei extraction
15. Decant the supernatant into the waste beaker and add 1 mL of ice-cold 1XHB to the pellet per gram of tissue being processed (e.g. 3 mL for 3 g tissue).
SECTION: Crude nuclei extraction
16. Resuspend the pellet using a fresh paint brush pre-soaked in ice-cold 1XHB. Mix with a P1000 pipette using a cut-tip until the solution is homogeneous.
SECTION: Crude nuclei extraction
17. Transfer each mL of the nuclei suspension into a fresh 2 mL Protein LoBind microcentrifuge tube (equivalent to 1 g tissue/tube).
SECTION: Crude nuclei extraction
18. Spin the 2 mL tubes for 5 min at 7,000 x g at room temperature.