TITLE: Sanger Tree of Life HMW DNA Extraction: Modified Omega Bio-Tek E.Z.N.A.®
AUTHORS: Amy Denton, Caroline Howard

[DESCRIPTION]
This protocol describes the manual extraction and SPRI of HMW DNA from fresh frozen jellyfish samples intended for long-read sequencing using a combination of the Omega Bio-Tek E.Z.N.A.® Mollusc & Insect DNA kit and Sera-Mag™ SpeedBeads. It  is effective for approximately 50 to 60% of jellyfish species covered by the Tree of Life Programme for the Aquatic Symbiosis Genomics (ASG) project. This protocol has resulted in successful extractions for a number of species including Mastigias papua, Mastigias albipunctatus, Rhopilema esculentum, Catostylus mosaicus, Cassiopea ornata, Cassiopea xamachana, Mertensia ovum, Bolinopsis mikado, Pseudorhiza haeckeli, Aurelia spp. and Pelagia noctiluca.

The output of this protocol is HMW DNA, which depending upon yield and genome size of the species, can be directed towards either HMW DNA Fragmentation: Diagenode Megaruptor®3 for LI PacBio or HMW DNA Fragmentation: g-Tube for ULI PacBio. 

This protocol was developed through R&D by the Tree of Life Core Laboratory; it was primarily adapted from the Omega Bio-Tek E.Z.N.A.® Mollusc & Insect DNA kit protocol by combining the initial lysis and precipitation (steps 1 to 10) with reduced incubation temperatures, omission of vortexing and the addition of a bead-based extraction using Sera-Mag™ SpeedBeads, as utilised in the Sanger Tree of Life HMW DNA Extraction: Automated Plant Organic HMW gDNA Extraction (POE) protocol.


Acronyms
HMW: high molecular weight
LI: low input
ULI: ultra-low input

[BEFORE_START]
Set a heat block to 25 °C.
Ensure that an adequate amount of Sera-Mag™ SpeedBead solution has been prepared prior to initiating the protocol. A volume of 600 to 800 μL is needed per sample, and requires 50% PEG 8000, 10% Tween-20, SpeedBead wash suspension and SpeedBead binding solution to be prepared prior to initiating the protocol (details in the Materials section).
Remove the prepared Sera-Mag™ SpeedBead solution from the fridge 30 minutes before starting the extraction step so they can be brought to room temperature.
Remove the AMPure PB beads from the fridge 30 minutes before starting the 0.45X SPRI step to allow them to be brought to room temperature. 

[GUIDELINES]
Input amounts of 100 to 200 mg of fresh frozen jellyfish tissue are required for this protocol. Smaller input amounts can be used, however the yields of DNA obtained may be lower.
Ideally fresh frozen samples should be used for this protocol. Ethanol preserved samples can also be processed using this protocol, however the yields of DNA obtained may be lower.
Keep samples on dry ice until the lysis buffer is ready to be added to them to maintain temperature and prevent nucleic acid degradation.
An experienced operator can expect to comfortably process 8 samples, with approximately 3 to 4 hours handling time over a start to finish period of 4 to 5 hours. This estimation excludes 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]
SECTION: Sample Lysis &amp; Precipitation
1. Set a heat block to 25 °C.
SECTION: Extraction
16. Add 600 to 800 μL Sera-Mag™ SpeedBead solution to each sample - the Sera-Mag™ SpeedBead solution should be vortexed before use to ensure that the beads are resuspended.
SECTION: Sample Lysis &amp; Precipitation
2. Label 1.5 mL BioMasher tubes for each sample being processed. Save the BioMasher pestles for later use in their sterile packet, also labelling them for each sample.
SECTION: Sample Lysis &amp; Precipitation
3. Transfer the samples into their corresponding 1.5 mL BioMasher tube.
SECTION: Sample Lysis &amp; Precipitation
4. Add 350 μL of ML1 buffer and 10 μL Proteinase K solution to each sample.
SECTION: Sample Lysis &amp; Precipitation
5. Use the BioMasher pestle to manually disrupt the sample within the buffer. Make sure to retain these pestles for further sample disruption at a later stage by returning them to their labelled sterile packets.
SECTION: Sample Lysis &amp; Precipitation
6. Incubate samples for 15 minutes at 25 °C.
SECTION: Sample Lysis &amp; Precipitation
7. Halfway through incubation, return to the samples and manually disrupt any tissue using the BioMasher pestles.
SECTION: Sample Lysis &amp; Precipitation
8. After incubation, transfer samples from the 1.5 mL BioMasher tubes to fresh 2 mL microcentrifuge tubes using a wide bore pipette – label these 2 mL microcentrifuge tubes for each sample.
SECTION: Sample Lysis &amp; Precipitation
9. Transfer samples to the fume hood for chloroform separation, then add 350 μL of chloroform:isoamyl (24:1) to each sample.
SECTION: Sample Lysis &amp; Precipitation
10. Transfer samples to the HulaMixer™, rotating at 8 rpm for 5 minutes.
SECTION: Sample Lysis &amp; Precipitation
12. Return samples to the fume hood in order to transfer the upper aqueous phase to a clean 2 mL microcentrifuge tube using a wide bore pipette tip. Avoid transferring the milky interphase containing contaminants and inhibitors.
SECTION: Sample Lysis &amp; Precipitation
13. Add 1 X volume of BL buffer and 5 μL RNase A to each sample. For example, if 400 μL aqueous upper phase was recovered in Step 12, add 400 μL of BL buffer. Pipette mix 15 times with a wide bore pipette tip.
SECTION: Sample Lysis &amp; Precipitation
14. Incubate samples for 10 minutes at 25 °C.
SECTION: Sample Lysis &amp; Precipitation
15. Add 1 x volume 100% ethanol: for example, if 400 μL aqueous upper phase was recovered in Step 12, add 400 μL of 100% ethanol. Transfer samples to the HulaMixer, rotating at 8 rpm for 5 minutes. 
SECTION: Extraction
17. Invert the sample tubes 10 to 20 times to ensure that beads are suspended in the lysate, then allow at least 5 minutes for binding.
SECTION: Extraction
18. Briefly centrifuge the sample in a mini-centrifuge to collect at the bottom of the tubes.
SECTION: Extraction
19. Place the tubes on the magnetic rack and allow 2 to 5 minutes for the beads to migrate. Be aware that more viscous samples will take longer. Remove the supernatant and discard.
SECTION: Extraction
20. Add 1 mL 80% ethanol opposite the bead pellet, incubate for 30 seconds, then aspirate and discard.
SECTION: Extraction
21. Repeat for a total of two washes.
SECTION: Extraction
22. Remove the tubes from the magnetic rack and add 200 μL Buffer AE directly to the beads. Mix 15 times using a wide bore pipette tip in order to dislodge the beads from the side of the tube.
SECTION: Extraction
23. Incubate samples for 15 minutes at room temperature, with a gentle mix halfway through and again at the end.
SECTION: Extraction
24. Briefly centrifuge the samples in a mini-centrifuge and place on a magnetic rack, allowing 2 to 5 minutes for bead migration.
SECTION: Sample Lysis &amp; Precipitation
11. Centrifuge samples at 10,000 g for 2 minutes at room temperature. The samples will then have three distinct phases as seen below in Figure 1: an upper aqueous phase containing the gDNA, a milky interphase containing inhibitors and contaminants, and a lower organic phase containing denatured proteins.

 

SECTION: Extraction
25. Using a 200 μL wide bore pipette tip, carefully transfer the supernatants containing purified gDNA to fresh 1.5 mL microcentrifuge tubes.
SECTION: Extraction
26. Remove samples from the magnetic rack and add 200 μL Buffer AE directly to the bead pellet. Incubate samples on the heat block for 3 minutes at 25 °C, shaking at 1,000 rpm. 
SECTION: Extraction
27. Centrifuge samples briefly in a mini-centrifuge and place on a magnetic rack, allowing 2 to 5 minutes for bead migration.
SECTION: Extraction
28. Using a wide bore pipette tip, carefully transfer the supernatants containing purified gDNA to the same 1.5 mL microcentrifuge tubes as in Step 25. Pipette-mix 5 to 10 times to homogenise the gDNA. The total eluate volume for each sample will now be 400 μL.

SECTION: 0.45X SPRI
29. Add 0.45X AMPure PB beads to the eluates (180 μL of beads for 400 μL eluate). Mix 15 times with a wide bore pipette tip.
SECTION: 0.45X SPRI
30. Incubate samples for 5 minutes at room temperature.
SECTION: 0.45X SPRI
31. Centrifuge samples in a mini-centrifuge for 1 to 2 seconds, then place on a magnetic rack. Allow 2 to 5 minutes for the beads to migrate.
SECTION: 0.45X SPRI
32. Remove and discard the supernatant.
SECTION: 0.45X SPRI
33. Keep samples on the magnetic rack and add 1 mL 80% ethanol. Incubate for 30 seconds and then remove and discard.
SECTION: 0.45X SPRI
34. Repeat for a total of two 80% ethanol washes.
SECTION: 0.45X SPRI
35. Remove samples from the magnetic rack and add 130 μL EB Buffer. Pipette mix 15 times with a wide bore pipette.
SECTION: 0.45X SPRI
36. Incubate samples on a heat block for 15 minutes at 37 °C.
SECTION: 0.45X SPRI
37. Return samples to the magnetic rack and transfer the supernatant containing the purified and SPRI'ed gDNA into a new 1.5 mL microcentrifuge tube.
SECTION: 0.45X SPRI
38. Incubate the DNA at room temperature overnight and perform the required QC the following morning.
SECTION: 0.45X SPRI
39. Store the DNA at 4 °C.