Sanger Tree of Life Sample Homogenisation: Cryogenic Bead Beating of Plants with FastPrep-96
Caroline Howard, Benjamin Jackson
Plant tissue disruption
cryogenic bead beating
tissue homogenisation
bead bashing
tissue interruption
cryofracture
plant tissue preservation.
Abstract
Disruption under cryogenic conditions is vital to interrupt the often tough, flexible and rigid structure of plant tissues whilst maintaining nucleic acid integrity. Cryogenic bead beating on the FastPrep-96, using 3 × 3 mm stainless steel beads in 1.9 mL Tri-coded FluidX tube, allows for the rapid disruption of up to 96 plant samples simultaneously. Further, the minimal handling time per sample and closed environment of the tube during disruption results in minimal risk of ambient temperature exposure, sample loss or cross contamination.
We recommend an input mass of 10–100 mg flash-frozen plant tissue, dissected into <1 cm2 pieces. Input requirements will vary depending on the downstream protocol to be performed, the plant species used, tissue type and sample quality. High-quality, young leaf material should be preferentially selected for optimal outcomes with ToL’s downstream applications; alternative tissue types can be selected (e.g. herbaceous stem, petiole or flower), but outcomes may vary. Non-pliable, rigid or highly fibrous tissues (e.g. woody or rigid, fibrous stem) should be avoided for optimal disruption outcomes, however if a necessity, these structures should be preemptively interrupted and dissected into pieces smaller than 5 mm2. Modifications to bead type and number of disruptions can be made to accommodate alternative sample types.
Following disruption, prepped tissues can be used immediately for appropriate downstream procedures, or can be stored long term at –70 °C with few detrimental effects. Approved downstream procedures of beat beaten plant tissue includes: HMW gDNA extraction for long-read PacBio HiFi sequencing, RNA extraction for RNA-seq and Hi-C genomic analysis.
Acronym HMW: high molecular weightgDNA: genomic DNAHiFi: high fidelity
Steps
Laboratory protocol
Prepare all necessary equipment prior to starting and place any applicable items (e.g. cold blocks, tools) onto dry ice.
Add 3 x 3 mm sterile stainless steel beads (Qiagen PN 69997) to the required number of 1.9 mL Tri-coded FluidX tubes and place into a cold block on dry ice. Alternatively, beads can be added after the sample as long as they have been pre-chilled.
Weigh 10–100 mg of the selected whole-frozen tissue on dry ice, dissecting into <1 cm2 pieces if required, and transfer into a prechilled 1.9 mL FluidX tube containing beads.
- Whole-frozen tissue can be dissected to size with scissors or a scalpel, or fragmented by crushing with an appropriate tool.
- Ensure tissues remain under cryogenic conditions at all times to avoid freeze/thaw cycles.
- Recalcitrant tissue types (see ‘guidelines’), should be dissected into pieces smaller than 5 mm2, and structures should be manually interrupted by crushing.
Repeat step 3 for each sample, and transfer all to a FluidX 48-rack on dry ice.
- Up to 96 samples can be processed simultaneously over 2 FluidX 48-racks.
- If only one FluidX 48-rack is used, a second rack containing an equal number of empty 1.9 ml Tri-coded FluidX tubes should be prepared to balance the FastPrep-96 during operation.
Submerge the FluidX 48-rack/s containing samples in liquid nitrogen (LN2) until temperature equilibrates (bubbling stops, ~15 seconds).
- Ensure the appropriate PPE is worn when handling, operating near or decanting LN2, see ‘Health & Safety warnings’.
Remove the FluidX 48-rack/s containing samples from the LN2 with tongs, allow any remaining LN2 to drain, and place onto the FastPrep-96.
- Samples should be handled efficiently once removed from the LN2 to limit exposure to ambient temperature.
Homogenise the frozen samples on the FastPrep-96 for 30 seconds at 1,600 rpm.
Remove the 48-rack/s containing samples from the fastPrep-96 and resubmerge in LN2 until temperature equilibrates (bubbling stop, ~15 seconds).
Repeat steps 6 to 8 twice, for a total of 3 disruptions, until tissues are completely disrupted into a fine powder.
- LN2 submersion is not necessary following the final disruption, but may be performed to avoid samples thawing.
- Manual inspection of samples is recommended for novel specimens; if any whole frozen tissue remains, steps 6 to 8 can be repeated up to a total of 6 times. After 6 disruptions, using an alternative specimen and/or tissue type or alternative bead type (e.g. 2 x 3 mm tungsten carbide beads) is recommended.
Place the FluidX 48-rack/s containing samples onto dry ice for >3 minutes to ensure all disrupted tissue remains frozen.
Proceed to the required downstream procedure, alternatively, disrupted plant tissue can now be stored long term at –70 °C with little detrimental effects observed.* Disrupted plant tissues in cryogenic storage should not be thawed at any time prior to its immediate use in downstream applications.
