Trekker protocol videos
See how it works! These Trekker protocol videos demonstrate key steps in the Trekker workflow to generate spatially tagged, isolated single nuclei from fresh-frozen tissue samples.
Please note that these videos only apply to Trekker assays compatible with fresh-frozen tissue.
Tissue melting on tile: Precise placement method
Sample is placed onto a tile with precise alignment. This method is recommended if the sample is significantly larger or smaller than the tile.
Tissue melting on tile: Stamping method
Sample is stamped onto a tile. This method is recommended if the sample is the same size as the tile and does not require precise alignment.
TIPS
- To flatten the sample after sectioning, use a brush to gently press the sample section into the cold block. Turning the sample section over can also reduce the curling of the OCT (optimal cutting temperature) compound.
- After melting the sample onto the tile, immediately proceed to process the sample or store for later use. DO NOT place the tile with the melted sample back on the cryostat. This will cause the sample to refreeze, which may reduce downstream data quality for some tissues.
- To store for later processing, freeze the slide with the tile and melted sample on dry ice, place it in a slide container, and store it at –80°C.
TIPS
- To flatten the sample after sectioning, use a brush to gently press the sample section into the cold block. Turning the sample section over can also reduce the curling of the OCT (optimal cutting temperature) compound.
- If needed after applying the stamping method, add a finger underneath the tile to further melt the sample onto the tile.
- After melting the sample onto the tile, immediately proceed to process the sample or store for later use. DO NOT place the tile with the melted sample back on the cryostat. This will cause the sample to refreeze, which may reduce downstream data quality for some tissues.
- To store for later processing, freeze the slide with the tile and melted sample on dry ice, place it in a slide container, and store it at –80°C.
Tissue staining and UV cleavage
Trekker 3x3 tile with melted sample is moved to a 12-well plate for UV cleavage and incubation. For frozen samples, thaw your tile before this step.
TIPS
- After adding UV Cleavage Buffer, there is a 15 min window before the next step. This is a good time to move your sample as needed, e.g., away from the cryostat and to your bench.
- If you are processing multiple samples, keep as much distance as possible between wells to avoid bleed-over effects of UV light from neighboring wells.
- The 7.5 min incubation after UV exposure allows the oligos to diffuse from the tile into the sample tissue. This incubation is another point when you can move samples from one location to another.
Tissue dissociation from Trekker tile
Sample tissue is dissociated from the Trekker tile after UV exposure and a 7.5 min incubation.
TIPS
- If needed, once 1 ml of Buffer A has been added, reduce the volume of your pipette to ~180 µl and press to the first stop to minimize the production of bubbles.
Tissue trituration
Trekker tile is removed and the sample is broken up further through trituration.
TIPS
- When gripping the tile, hold the clear, outer section of the tile. Avoid scratching the inner, white portion of the tile to prevent bead contamination.
- When placing the tile in a neighboring well, it may be useful to check the tile to see how much tissue was left behind, or to take a photograph as a record of the thoroughness of sample dissociation.
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