TITLE: High Molecular Weight DNA Extraction from Recalcitrant Plant Species for Third Generation Sequencing
AUTHORS: Rachael Workman, Renee Fedak , Duncan Kilburn, Stephanie Hao, Kelvin Liu, Winston Timp

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">Single molecule sequencing requires optimized sample and library preparation protocols to obtain long-read lengths and high sequencing yields. Numerous protocols exist for the extraction of DNA from plant species, but the genomic DNA from these extractions is either too low yield, of insufficient purity for sensitive sequencing platforms, e.g. nanopore sequencing, too fragmented to achieve long reads, or otherwise unattainable from recalcitrant adult tissue. This renders many plant sequencing projects cost prohibitive or methodologically intractable. Existing protocols are also labor intensive, taking days to complete. Our protocol described here yields micrograms of high molecular weight gDNA from a single gram of adult or seedling leaf tissue in only a few hours, and produces high quality sequencing libraries for the Oxford Nanopore system, with typical yields ranging from 3-10 Gb per R9.4.1 flowcell and producing reads averaging 5-8 kb, with read length N50s ranging from 6-30 kb depending on the style of library preparation (details in sequencing outcomes section), and maximum lengths extending up to 200 kb+.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div></div>

[STEPS]
?. [Nuclei Isolation]
Grind  of tissue, preferably fresh or snap frozen, into fine powder in liquid nitrogen with a mortar and pestle. Immediately transfer ground tissue to capped 250 mL bottle containing  NIB. Cap bottle and attach to end over end mixer, rotating at max speed for  at .
1 g
10 ml
4 °C
?. [Nuclei Isolation]
Grind  of tissue, preferably fresh or snap frozen, into fine powder in liquid nitrogen with a mortar and pestle.
1 g
?. [Nuclei Isolation]
Immediately transfer ground tissue to capped 250 mL bottle containing  NIB.
10 ml
?. [Nuclei Isolation]
Using a funnel, gravity filter homogenate through 5 layers of Miracloth into a 50 mL conical tube. Cap tube and centrifuge at  for .
4 °C
?. [Nuclei Isolation]
Using a funnel, gravity filter homogenate through 5 layers of Miracloth into a 50 mL conical tube.
?. [Nuclei Isolation]
Decant the supernatant and add  cold NIB to pellet. Resuspend pellet with paint brush pre-soaked in NIB.
1 ml
?. [Nuclei Isolation]
Decant the supernatant and add  cold NIB to pellet.
1 ml
?. [Nuclei Isolation]
Transfer  nuclei suspension to a 15 mL conical.
1 ml
?. [Nuclei Isolation]
Bring volume up to  with ice-cold NIB. Centrifuge at  for .
15 ml
4 °C
?. [Nuclei Isolation]
If after centrifugation supernatant is clear, decant supernatant and take pellet into step 7. If coloration remains in the supernatant after centrifugation, dispose of supernatant and resuspend nuclei pellet in NIB, bringing volume up  - , and  .
10 ml
15 ml
For conifer tissue it was important to repeat this step 3-5X; however, in maize tissue subsequent washes have been unnecessary.
?. [Nuclei Isolation]
Bring volume up to  with ice-cold NIB.
15 ml
?. [Nuclei Isolation]
Remove supernatant and resuspend in  1X HB.
1 ml
?. [Nuclei Isolation]
At this point, you can either snap freeze nuclei or proceed to lysis.
?. [Snap Freezing]
Spin down your nuclei suspension in a 1.5 mL tube at  -  for , remove supernatant and snap freeze in liquid nitrogen, then store at .
-80 °C
?. [Lysis Preperation]
Spin down your nuclei suspension in a 1.5 mL tube at  -  for , remove supernatant, and proceed to lysis.
?. [Nanobind-assisted DNA Purification]
Resuspend isolated plant cell nuclei with  of Proteinase K. Vortex on high until fully resuspended. Spin tube on mini-centrifuge for  to remove liquid from cap.
30 µl
?. [Nanobind-assisted DNA Purification]
Resuspend isolated plant cell nuclei with  of Proteinase K.
30 µl
?. [Nanobind-assisted DNA Purification]
Optional for removal of RNA: add  of RNase A and pulse vortex 5X to mix. Spin tube on mini-centrifuge for to remove liquid from cap. Incubate at  for .
10 µl
0 Room temperature
?. [Nanobind-assisted DNA Purification]
Add  of RNase A and pulse vortex 5X to mix
10 µl
?. [Nanobind-assisted DNA Purification]
Add  Buffer PL1 to the sample and pulse vortex 10X to mix. Spin tube on mini-centrifuge for  to remove liquid from cap.
80 µl
● Thorough mixing at this step is necessary to ensure complete lysis of the nuclei. Sample should appear cloudy and very viscous but homogeneous.● If sample appears inhomogeneous, vortex continuously on high speed for  . DNA is protected at this stage and vortexing should not damage the DNA significantly.
?. [Nanobind-assisted DNA Purification]
Incubate on a ThermoMixer at  and 900 rpm for .
55 °C
● Mid-lysis pulse vortex (5X) will enhance mixing (pulse vortexing at this stage will stillresult in very large DNA).● If a ThermoMixer is not available, incubate at  in a dry bath incubator or waterbath. Invert mix 5X every  .● If sample does not appear to be lysed, incubation time can be increased up to  .
?. [Nanobind-assisted DNA Purification]
Centrifuge lysate for  at  at .
0 Room temperature
?. [Nanobind-assisted DNA Purification]
Transfer supernatant to new 1.5 mL Protein LoBind microcentrifuge tube using a wide bore pipette.
The DNA should be handled carefully from this point forward to prevent shearing.
?. [Nanobind-assisted DNA Purification]
Add Nanobind disk to supernatant followed by 1X volume of isopropanol.
● If the volume of supernatant is greater than 150 µL, the volume of isopropanol should be adjusted accordingly.● For best results, the Nanobind disk should always be added before isopropanol.
?. [Nanobind-assisted DNA Purification]
Carefully mix for  at . Verify that the fluid is continuously mixing and that theNanobind disk remains submerged in the binding solution throughout the binding process for efficient recovery of DNA.
0 Room temperature
● Mixing can be performed by inverting the tubes and placing them cap-side downon a HulaMixer (Thermo Fisher) or similar device. ● If a HulaMixer in not available, manually mix the tubes by inversion (e.g., 5X inversions every ) to facilitate binding.● A tube rotator is not recommended, as low reaction volume precludes adequate mixing during end over end rotation.
?. [Nanobind-assisted DNA Purification]
Place tubes in magnetic tube rack.
?. [Nanobind-assisted DNA Purification]
While the tube is on the magnet, discard the supernatant, avoiding contact with the Nanobind disk.
?. [Nanobind-assisted DNA Purification]
Add  of Buffer PW1, inversion mix 4X, replace tube rack on the magnetic base, and discard the supernatant. (1/2)
500 µl
?. [Nanobind-assisted DNA Purification]
Add  of Buffer PW1, inversion mix 4X, replace tube rack on the magnetic base, and discard the supernatant. (2/2)
500 µl
?. [Nanobind-assisted DNA Purification]
Remove any residual liquid from cap of 1.5 mL tube.
?. [Nanobind-assisted DNA Purification]
Spin the tube on a mini-centrifuge for  and remove the residual liquid.
If the Nanobind disk is blocking the bottom of the tube, gently push it aside with the tip of the pipette. At this stage, DNA is tightly bound to the disk and gently manipulating the disk with a pipette tip should not cause any damage.
?. [Nanobind-assisted DNA Purification]
Repeat step 23.   If residual wash buffer remains on Nanobind or sidewalls of tube, repeat step 23 until tube and Nanobind appears dry.
?. [Nanobind-assisted DNA Purification]
Add  to  Buffer EB spin the tube on a mini-centrifuge for . Incubate at  for . Confirm the entire Nanobind disk is fully immersed in Buffer EB during elution.
50 µl
200 µl
0 Room temperature
?. [Nanobind-assisted DNA Purification]
Add  to  Buffer EB spin the tube on a mini-centrifuge for .
50 µl
200 µl
?. [Nanobind-assisted DNA Purification]
Collect extracted DNA by transferring eluate to a new 1.5 mL microcentrifuge tube using a wide bore pipette. Spin the tube on a mini-centrifuge for  and transfer any remaining eluted DNA. Repeat if necessary.
● Using a wide bore pipette helps to ensure that the extracted DNA is 50–300+ kb in length.● For high molecular weight DNA, the extracted DNA can be inhomogeneous. This is normal and is one of the challenges with working with high molecular weight DNA. Leaving the extracted DNA at room temperature overnight can increase the homogeneity.
?. [Nanobind-assisted DNA Purification]
Analyze the recovery and purity of the DNA by NanoDrop and Qubit.
?. [Nuclei Isolation]
Cap bottle and attach to end over end mixer, rotating at max speed for  at .
4 °C
Alternatively, lay bottles on their side on a shaker (150 rpm), or transfer ground tissue to a beaker capped with foil and mix on stir plate with stir bar at   for  .
?. [Nuclei Isolation]
Cap tube and centrifuge at  for .
4 °C
Speed of centrifugation is dependent upon size of genome: For larger genomes such as redwood (30 Gb), we spun down at  .For smaller genomes like walnut (1 Gb) we spun down at  .For maize (2.5 Gb) was spun down at  .
?. [Nuclei Isolation]
Resuspend pellet with paint brush pre-soaked in NIB.
Pipetting up and down with wide bore tips works for some species (for example maize) but for many species the pellet is too sticky to allow for sufficient resuspension by pipet.
?. [Nuclei Isolation]
Centrifuge at  for .
4 °C
?. [Nanobind-assisted DNA Purification]
Vortex on high until fully resuspended. Spin tube on mini-centrifuge for  to remove liquid from cap.
● Proper resuspension and mixing of the nuclei pellet is necessary to ensure efficient lysis.● Vortexing is critical and will not damage high molecular weight DNA at this step.● If removal of RNA is not necessary, proceed directly to step 12.
?. [Nanobind-assisted DNA Purification]
Spin tube on mini-centrifuge for to remove liquid from cap. Incubate at  for .
0 Room temperature
?. [Nanobind-assisted DNA Purification]
Incubate at  for . Confirm the entire Nanobind disk is fully immersed in Buffer EB during elution.
0 Room temperature
● Elution volumes can be adjusted depending on downstream concentrationrequirements.● The Nanobind disk may be compressed into the bottom of the tube using apipette tip without causing any damage to the process.