Sanger Tree of Life Sample Homogenisation: Covaris cryoPREP® Automated Dry Pulverizer
Naomi Park, Caroline Howard, Juan Pablo Narváez-Gómez, Haddijatou Mbye, graeme oatley, Michelle Strickland
sample homogenisation
cryoPREP
tissue disruption
reference genome
long read sequencing
cryogenic
cryogenic tissue disruption
Abstract
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.
Steps
Transfer sample to TissueTUBE
Place the sample into the tissueTUBE TT1 and seal using the adapter and attaching the sample tube on the top.
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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.
Disruption
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”.
Hold the sample tube such that the tissue remains in the TissueTUBE bag.
Loosen the tube by ¼ to ½ turn to allow the air inside the tube to escape during disruption.
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.
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.
Insert the TissueTUBE assembly into the cryoPREP and close the lid.
Check cryoPREP settings and press the green “Activate” button. For recommended disruption settings, check Table 1.
Place the bag in liquid nitrogen (as per step 4) before and after each impact.
Additional impacts can be performed if the sample is not fully disrupted and the TissueTUBE bag remains intact.
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.
Immediately put the inverted TissueTUBE assembly in dry ice or a cold rack if available.
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.
Decontaminate adapters for reuse
Prepare an appropriate quantity of Virkon disinfectant or alternative, following manufacturer’s instructions.
Dispose of the TissueTUBE bag as biological waste.
Check the adapter for large pieces of material. If material is present, remove with an Azowipe or forceps and dispose of as biological waste.
Place adapters in disinfectant solution and leave overnight.
Dispose of solution following local guidance.
Rinse adapters thoroughly with ultrapure sterile water.
Allow adapters to dry in a sterile environment, under UV light if possible.
