TITLE: Sanger Tree of Life Sample Homogenisation:  Covaris cryoPREP® Automated Dry Pulverizer
AUTHORS: Juan Pablo Narváez-Gómez, Haddijatou Mbye, graeme oatley, Michelle Strickland, Caroline Howard

[DESCRIPTION]
This protocol describes the procedure for cryogenic homogenisation of tissue samples using the Covaris cryoPREP® Automated Dry Pulverizer CP02. The protocol is designed for DNA and/or RNA extraction aimed at long-read sequencing or RNA-Seq applications. The method is particularly effective for disrupting tissues with a mass exceeding 25 mg across all taxonomic groups studied in the Tree of Life Programme. However, it is not advisable for samples smaller than this, due to inherent tissue loss during processing. Additionally, the technique has limitations when applied to fibrous plant tissues and samples rich in polysaccharides (e.g., macroalgae and molluscs), where it may result in either insufficient disruption or excessive tissue loss. The resultant sample is compatible with any Sanger Tree of Life DNA and RNA extraction protocols as well as Hi-C library preparation.

[BEFORE_START]
Ensure an adequate amount of sample has been prepared according to weight recommendations provided in Table 2 of the Guidelines section.
Decant a suitable amount of liquid nitrogen into the liquid nitrogen Dewar flask for the number of samples being processed.

[GUIDELINES]
This method is not recommended for samples with mass less than 25 mg.
It is not recommended to cryoPREP samples >200 mg if preparing for multiple downstream procedures, as tissue disruption may be incomplete.
Expect to lose up to 15 mg of tissue. Disrupted samples sometimes may become trapped in the TissueTUBE bag corners, or adhere to the inside surface.
Samples must remain as cold as possible during disruption without introducing liquidised gas into the TissueTUBE. 
Place into liquid nitrogen immediately prior to and following each impact. Dry ice is not sufficiently cold for this procedure.


Table 1: Suggested settings for cryoPREP disruption of samples 
   Sample typeInitial Setting 1 impactSubsequent impacts (Setting x number of impacts)AlgaeYesS6 x 2AnnelidsYesS4 x 1Arthropod YesS6 x 1PlantYesS6 x 6FungiYesS6 x 6ProtistYesS6 x 1SpongesYesS2 x 2 Soft tissue: Vertebrates, Molluscs and otherYesS4 x 1Hard Tissue: Corals and othersYesS6 x 6


Table 2: Tissue weight required for CryoPREP 
   Lab Work CategoryTissue weight required before CryoprepDNA ExtractionMetazoa: 30–40 mg Plants/Algae/Fungi: 60–70 mgProtist 55–100 mg DNA - Plant Organic Extraction (POE)Plants/Mosses 80–90 mg HiCMetazoa: 60–70 mg Plants/Algae/Fungi: 60–70 mgRNA ExtractionMetazoa: 60–70 mg Plants/Algae/Fungi: 60–70 mgProtist 55–100 mg 
Additional Notes
It is good practice to check the TissueTUBE bag after each impact to ensure the bag is not damaged, to prevent sample loss. If the TissueTUBE bag appears damaged during disruption, replace the damaged TissueTUBE bag.
Occasionally discs of compressed material can form during disruption; break these up using chilled forceps after each impact.
If the sample is very small, the smaller Covaris cryoPREP TissueTUBE can be used, for larger samples use Covaris cryoPREP TissueTUBE TT1 extra thick.
FluidX tubes are used throughout the Tree of Life program to track samples, custom adapters have been 3D printed (on-site) in order to pair them with Covaris TissueTUBE bags. 

[STEPS]
SECTION: Disruption
3. Power on the cryoPREP instrument. Ensure all doors to the room are closed and all room occupants are wearing ear defenders. Set the setting dial to “1”.
SECTION: Transfer sample to TissueTUBE
1. Place the sample into the tissueTUBE TT1 and seal using the adapter and attaching the sample tube on the top.

 
SECTION: Transfer sample to TissueTUBE
2. If processing multiple samples, bury the bag containing the sample in the dry ice whilst processing the others. Try to keep the samples as cold as possible.
SECTION: Disruption
4. Hold the sample tube such that the tissue remains in the TissueTUBE bag.
SECTION: Disruption
5. Loosen the tube by ¼ to ½ turn to allow the air inside the tube to escape during disruption.
SECTION: Disruption
6. Place the TissueTUBE assembly so the bag is immersed in liquid nitrogen and hold for 1 to 2 seconds after the boiling subsides. Do not submerge the collar or any part above to prevent liquid nitrogen entering the bag.
SECTION: Disruption
7. Remove the TissueTUBE assembly from the liquid nitrogen and check for liquid nitrogen or liquidised air inside the bag. Allow the gas to evaporate if present. 
SECTION: Disruption
8. Insert the TissueTUBE assembly into the cryoPREP and close the lid.
SECTION: Disruption
9. Check cryoPREP settings and press the green “Activate” button. For recommended disruption settings, check Table 1.
SECTION: Disruption
10. Place the bag in liquid nitrogen (as per step 4) before and after each impact.
SECTION: Disruption
11. Additional impacts can be performed if the sample is not fully disrupted and the TissueTUBE bag remains intact.
SECTION: Disruption
12. Tighten the adapter and submerge the TissueTUBE bag in liquid nitrogen one last time. Take the sample out and invert so the disrupted tissue leaves the bag and enters the sample tube, quickly and vigorously agitate/flick the bag to transfer the disrupted sample into the tube.
SECTION: Disruption
13. Immediately put the inverted TissueTUBE assembly in dry ice or a cold rack if available.
SECTION: Disruption
14. Remove the TissueTUBE bag and adapter and replace the lid on the tube. The disrupted tissue can be transferred to a new tube if required for downstream processing or if aliquots have to be made.
SECTION: Decontaminate adapters for reuse
15. Prepare an appropriate quantity of Virkon disinfectant or alternative, following manufacturer’s instructions.
SECTION: Decontaminate adapters for reuse
16. Dispose of the TissueTUBE bag as biological waste.
SECTION: Decontaminate adapters for reuse
17. Check the adapter for large pieces of material. If material is present, remove with an Azowipe or forceps and dispose of as biological waste. 
SECTION: Decontaminate adapters for reuse
18. Place adapters in disinfectant solution and leave overnight.
SECTION: Decontaminate adapters for reuse
19. Dispose of solution following local guidance.
SECTION: Decontaminate adapters for reuse
20. Rinse adapters thoroughly with ultrapure sterile water. 
SECTION: Decontaminate adapters for reuse
21. Allow adapters to dry in a sterile environment, under UV light if possible.