Nuclei extraction optimization

Extracting nuclei from tissue during sample preparation is a critical step for successful use of the Trekker Single‑Cell Spatial Mapping Kits. Optimizing this process is essential to obtain high-quality nuclei counts while minimizing damage and contamination.

Standard instructions for tissue preparation—including nuclei extraction—for use with Trekker kits are outlined in the relevant Trekker user manual (see Table 1 below). For further guidance, we have developed tissue-specific modified protocols for many commonly used tissue types (see Tables 1–2 below). Table 2 also includes example data we have collected during our experiments with these sample types. Please note that results may vary with each user’s samples. Further optimization may be necessary, either due to variability in sample collection and quality or for tissues not covered below. For additional guidance, refer to the “General Recommendations” section below. If needed, we recommend working closely with the Takara Bio Technical Support team (technical_support@takarabio.com) for help specific to your experiments.

For tissues like spleen or pancreas, which are expected to have high levels of RNase activity, we recommend increasing the amount of RNase Inhibitor in master mixes to 1.8X the stated concentration.

For plant tissue preparation recommendations, please reach out to the Takara Bio Technical Support team (technical_support@takarabio.com).

General recommendations

Consider these key aspects when optimizing nuclei extraction:

1. Tissue preparation and storage
   • Overall protocol: For guidelines regarding tissue harvesting, preservation, and cryostat sectioning, refer to the Trekker and Seeker Preserving and Sectioning Tissue Protocol-At-A-Glance.
   • Sample storage: We recommend storing frozen tissues embedded in OCT (optimal cutting temperature) compound to minimize tissue dehydration. For guidelines on OCT embedding, see the following example protocol.
   • Maintaining cold temperatures: Equilibrate the sample block in the cryostat at least 30 min before sectioning. Larger samples may require more time to fully equilibrate. While performing the Trekker protocol, keep tissue samples on ice throughout the entire extraction procedure to minimize degradation and nuclei lysis.
2. Grinding and lysis
   • Gentle grinding: For fibrous tissues—including diseased, muscle, and many types of tumor tissues—mechanical grinding may be required. This includes plastic micropestle grinders and automated tissue grinders. Of note, we have observed that glass Dounce homogenizers are harsh on tissue and may damage nuclei. For a stronger plastic pestle, consider using the Kimble Biomasher II Closed System Tissue Grinder (DWK Life Sciences, 749625-0010).
   • Lysis buffer optimization: While the buffers included in the Trekker kits work well for many tissues, you may choose to optimize the protocol using different buffers. The appropriate choice and concentration of lysis buffer are essential for efficiently lysing the cell membrane efficiency while preserving the integrity of the nuclear membrane.
   • Lysis timeline: Optimizing the duration of lysis incubation is important to minimize the risk of overlysis. During incubation, monitor the lysis effects for your nuclei preps to determine the total incubation time that delivers best results. For the lysis buffer provided with the Trekker kit, we recommend incubating samples for no longer than 10 min to prevent overlysis.
3. Minimizing debris and purifying nuclei
   • Filtration: After tissue homogenization, the lysate must be filtered through appropriately-sized cell strainers. We recommend the 20 µm cell strainers (see the Table 3 below) for most tissues, but up to 40 µm cell strainers may be appropriate for larger nuclei, such as those in liver or some types of tumor tissues. The Trekker protocol is optimized for minimal filtration, as this is a source of significant nuclei loss. Similarly, using non-recommended filters may lead to significant nuclei loss, as the filters commonly used for conventional nuclei isolation protocols are not optimized for the low sample input of the Trekker protocol.

     Name	Part number
     pluriStrainer Mini 20 μm (Cell Strainer)	43-10020-40
     pluriStrainer Mini 40 μm (Cell Strainer)	43-10040-40

     Table 3. Recommended cell strainers for use with the Trekker workflow.

   • Nuclei cleanup: While we recommend minimizing the amount of post-lysis washing, the recommended wash buffer exchanges and filtration steps are optimized for most mammalian tissues. If you observe significant debris (>20%) remaining, it may be necessary to perform additional wash and/or filtration steps. Gentle handling of nuclei is recommended to maintain nuclei health.
4. Nuclei counting and quality control
   • Fluorescent dyes for counting: Utilizing fluorescent dyes—such as Ethidium homodimer-1, AO/PI (Acridine orange and propidium iodide), or DAPI (4′,6-diamidino-2-phenylindole)—for nuclear staining improves counting accuracy by staining only nucleic acids and avoiding false counts from debris.
   • Cell counting: If your automated cell counter delivers unclear or inconsistent results, consider using a hemocytometer with a fluorescence microscope as an alternative counting method.
5. General best practices
   • Prepare materials in advance: Have all reagents and equipment ready and prechilled to the correct temperatures before starting the protocol to avoid delays.
   • Work quickly: Minimize the time taken for each step to reduce the risk of nuclei degradation and damage. Proceed to library preparation within 30 min (keeping the samples on ice) for best results.
   • Trial runs: If you are using a new tissue type, perform a trial run to optimize the protocol for your specific tissue type and ensure the quality of the extracted nuclei.
     • Use of the Trekker 10x10 Training Kit Bundle (SK020) or Trekker 10x10 Training Kit Bundle V2 (SK031) is highly recommended for optimizing sample preparation. The kit contains two training tiles and enough reagents for four reactions.
     • Initial optimization can be performed on tissue curls in microfuge tubes. For this purpose, excess OCT compound should be removed from the tissue before processing. Excess OCT compound will prevent nuclei dissociation by preventing isolation buffer from interacting with the sample tissue.
     • Once satisfactory results are obtained from a trial run, the protocol can be repeated once more with a training tile. If results are still good, the user should then move forward with their experiment.