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.
HMW: high molecular weight