TITLE: Sanger Tree of Life HMW DNA Extraction: Manual Mollusc Nanobind®
AUTHORS: Pacific Biosciences, Adam AB Bates, Caroline Howard

[DESCRIPTION]
This protocol describes the manual extraction of HMW DNA from mollusc samples intended for long-read sequencing using the Nanobind® tissue kit and following the ‘Extracting HMW DNA from black mystery snail tissue using Nanobind® kits’ procedure from PacBio. This process is effective for mollusc species covered by the Tree of Life Programme. The output of this protocol is high quality and quantity HMW DNA, which can be directed towards the HMW DNA Fragmentation: Diagenode Megaruptor®3 for LI PacBio protocol.


Acronyms
HMW: high molecular weight
LI: low input

[BEFORE_START]
Add 100% ethanol to the Buffers CW1 and CW2 as per manufacturer’s instructions.
Set a heat block to 55 °C.
Pre-chill the centrifuge to 4 °C.

[GUIDELINES]
This protocol uses the ‘Extracting HMW DNA from black mystery snail tissue using Nanobind® kits’ procedure, with the inclusion of the sample preparations, standard sample inputs and standard elution volumes used by Sanger Tree of Life.
This protocol works best with cryogenically disrupted material of around 30–50 mg input weight. If the tissue has not been cryogenically disrupted, place in a chilled petri dish and on ice, cut the tissue into smaller chunks, then powermash the sample in the CT buffer.
Before starting the protocol, keep samples on dry ice to maintain temperature and prevent nucleic acid degradation.
An experienced operator can expect to comfortably process 8 samples with a start to finish period of 4 hours. This estimation excludes overnight incubation at room temperature and subsequent QC checks.

Additional Notes:
FluidX tubes are used throughout the Tree of Life programme in order to track samples, therefore rather than the microcentrifuge tubes which have been mentioned in this protocol for DNA storage, all routine DNA extracts are stored in FluidX tubes.

[STEPS]
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1. Transfer tissue to a 2 mL microcentrifuge tube or a powermash tube if not cryoprepped and add 
750 μL ice cold buffer CT; powermashing if required.
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2. Centrifuge 6,000 × g at 4 °C for 5 minutes.
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3. Discard supernatant and add fresh 1 mL cold buffer CT.
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4. Pulse vortex 10 times for 1s (or mix with wide-bore tip) to dislodge pellet.
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5. Pellet homogenate by centrifuging at 6,000 × g at 4 °C for 5 minutes.
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6. Discard supernatant.
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7. Add 20 μL of Proteinase K to the pellet and pulse vortex 1s twice to dislodge the pellet.
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8. Add 150 μL of buffer CLE3 and pipette mix 10 times using a wide-bore tip.
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9. Incubate at 55 °C and 900 rpm for 30 minutes on the heat block.
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10. Spin on a mini-centrifuge to collect liquid from the lid.
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11. Add 20 μL of RNAse A.
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12. Incubate at 55 °C and 900 rpm on the heat block for another 30 minutes. This incubation can be longer, up to 2 hours, if there are still visible large tissue pieces remaining.
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13. Add 60μL buffer SB and pulse vortex 5 times to mix (wide-bore mixing is also acceptable).
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14. Centrifuge 10,000 × g at room temperature for 5 minutes.
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15. Transfer up to 300 μL of the supernatant to a new freshly labelled 2 mL microcentrifuge tube.
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16. Add 50 μL of buffer BL3 to the collected supernatant and inversion mix 10 times (this can also be done with a wide-bore tip).
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17. Spin on a mini-centrifuge to collect liquid from the lid.
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18. Add the Nanobind disk to the lysate.
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19. Add 350 μL isopropanol and inversion mix 10 times.
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20. Allow 15 minutes at room temperature for the DNA to bind to the disk.
This step should be performed on a platform rocker, alternatively, you could manually invert the tube every 3 minutes or place the sample on a HulaMixer at 25 rpm for 15 minutes with the reciprocal angle set at 50°.
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21. Place the tubes on the magnetic rack.
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22. Discard the supernatant, either using a pipette and taking care to avoid touching the disk or by tipping the supernatant out into a reservoir, ensuring that the disk and DNA are bound to the magnet.
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23. Add 500 μL of Buffer CW1, remove the tubes from the magnet and inversion mix 4 times, place back on the rack and discard the supernatant.
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24. Repeat step 23.
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25. Add 500 μL of Buffer CW2, remove the tubes from the magnet and inversion mix 4X, place back on the rack and discard the supernatant.
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26. Repeat step 25.
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27. Spin on a mini-centrifuge to collect liquid from the lid.
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28. Place on the magnetic rack and remove any residual supernatant.
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29. Add 75–100 μL Buffer EB directly to the nanobind disk and incubate for 10 min at room temperature.
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30. Collect supernatant using a wide-bore pipette tip.
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31. Allow 24 hours at room temperature for the DNA to relax before QC.