Seeker FAQs

The Seeker spatial transcriptomics kit is the first high-resolution, whole-transcriptome, spatial mapping kit enabling researchers to map and analyze the spatial gene expression of tens to hundreds of thousands of cells on a single tile.

The spatial resolution is 10 µm. This is achieved via 10 µm beads that are tightly packed together on the tile.

No. You only need the standard tools found in a typical molecular biology lab, such as heat blocks, thermal cyclers, and access to an Illumina sequencer.

The Seeker workflow takes about 8 hr from tissue melting through library prep. The actual hands-on time is about 2–3 hr.

There are two tile sizes:

• 3 mm x 3 mm
• 10 mm x 10 mm

Fresh-frozen samples are compatible with the Seeker kit.

The kit is compatible with tissues from all species without any further optimization of the protocol (i.e., no permeabilization optimization). This is because the assay involves neither tissue fixation nor tissue permeabilization.

Tissue sections for the Seeker tile should be 10 µm thick.

The CryoCube section (30 or 60 µm) is melted on top of the 10 µm tissue section after it is melted onto the tile.

The CryoCube overlay improves this assay in three different ways:

1. It keeps the tissue attached to the tile.
2. It prevents the tissue section from drying out. An exposed section runs the risk of drying out, which could make the tissue harder to remove from the tile.
3. It reduces lateral diffusion of mRNA. Failure to use the CryoCube covering may lead to more noise in the data and to mRNA leaking outside of the tissue region, especially around the borders of the tissue or air pockets (e.g., lung alveolar sacs).

For these reasons, the CryoCube overlay step is essential to achieve high-quality data.

As more customers run the Seeker kit with various plant tissues, we will update this FAQs page with any additional information, but the following tips help increase the chance of success.

• Make sure to start dissociation soon after tissue sectioning (i.e., within 1–2 hr after sectioning, without freezing the tile). Freezing the tile and tissue together (between Steps 12 and 13 of Section B on the protocol) and keeping it at –80°C for a couple of days may make it very difficult to dissociate.
• Incubate the tile with tissue clearing reagents for 60 min instead of 30 min.

The standard Seeker kit is optimized for processing mouse heart tissue. For human heart tissue, we recommend trying the following adjustments to the standard protocol:

• Increase hybridization time to 30 min.
• Increase reverse transcription time to 90 min.
• Run 13 cycles of PCR in the second stage of cDNA amplification.

Measure the concentration by Qubit quantification. If it is too low, re-amplify the cDNA.

1. Create a cDNA master mix according to the table below.

   Component	1 reaction
   cDNA Amp Buffer	25 μl
   cDNA Amp Primer Mix	2 μl
   cDNA Amp Enzyme	1 μl
   Nuclease-Free Water	22 μl–(X μl of cDNA) Example: 22 μl – 5 μl cDNA = 17 μl Nuclease-Free Water
   cDNA input	All remaining cDNA (recommended)
   Total	50 μl

2. Mix well by pipetting.
3. Briefly spin down the PCR tube.
4. Run the following PCR program.

   Temperature	Time	Cycles
   98ºC	2 min	3–6 cycles*
   98ºC	20 sec
   67ºC	20 sec
   72ºC	3 min
   72ºC	5 min
   4ºC	Hold

   Choose the number of cycles most appropriate for your sample (e.g., if you have very little cDNA, run closer to 6 cycles).

5. Vortex the SPRI beads and add 30 µl to each of the tubes (0.6-fold volume of the reaction mixture).
6. Vortex to mix for 10–15 sec.
7. Incubate at room temperature for 5 min.
8. Briefly centrifuge the tubes and place the tubes on the magnetic rack. Once the solution is clear, carefully remove and discard the supernatant.
9. Keeping the tubes on the magnetic stand, add 200 µl of 80% ethanol.
10. Wait 30 sec, then remove and discard the supernatant.
11. Add 200 µl of 80% ethanol.
12. Wait 30 sec, then remove and discard the supernatant.
13. Briefly spin the tubes to consolidate the remaining ethanol.
14. Place the tubes back on the magnetic rack, then remove and discard the remaining ethanol.
15. Let the SPRI beads dry at room temperature until the beads appear matte (~1–2 min). Be careful not to overdry.
16. Remove the tubes from the magnetic rack and add 10 µl of Nuclease-Free Water to each tube to elute. Pipette the beads to mix well and incubate at room temperature for 1 min.
17. Place the tubes on a magnetic rack and incubate for 1 min.
18. Transfer the supernatant to new 0.2 ml PCR tube(s).
19. Quantify the cDNA products using Qubit 1X dsDNA High Sensitivity Assay Kit and a Bioanalyzer or TapeStation system, following the manufacturer’s guidelines.

There is no permeabilization step, so no optimization is required. This is one of the major advantages of Seeker technology. The Seeker kit works on any species or tissue without requiring changes to the protocol.

Sometimes, tissue with low RNA quality or very low cellular density can result in a final library TapeStation profile that includes a very long PCR-bubble product on the trace (see example below).

To fix this, you have two options:

• Option 1: Re-run the tagmentation reaction with fewer indexing PCR cycles. Run 8–9 cycles instead of the recommended 12 cycles. If this does not work, try Option 2 below.
• Option 2. Run a reconditioning PCR protocol using the library as template. See instructions below.

  Component	1 reaction
  Tagged library product	2.5 μl
  Index Primer F (from Seeker Dual Indexing Primer Kit v2, Cat. No. K006)	2.5 μl
  Index Primer R (from Seeker Dual Indexing Primer Kit v2, Cat. No. K006)	2.5 μl
  Nuclease-Free Water	10 μl
  Nextera® PCR Master Mix (NPM)	7.5 μl
  Total	25 μl

  1. Mix well by pipetting.
  2. Briefly spin down the PCR tube.
  3. Run the following PCR program.

     Temperature	Time	Cycles
     72ºC	3 min	3 cycles
     95ºC	30 sec
     95ºC	10 sec
     55ºC	30 sec
     72ºC	30 sec
     72ºC	5 min
     4ºC	Hold

  4. Proceed with the same library cleanup and quantification steps in Section V.I, "Library Cleanup and Quantification" in the Seeker 3x3 Bundle V1.1 User Manual.

You can easily perform this conversion using the Seeker Primary Analysis Pipeline.

The Seeker Primary Analysis Pipeline reconstructs a spatial map of gene expression from FASTQ files generated by sequencing the Seeker libraries. Additionally, it performs quality control to help users determine their next steps.

You can either install the analysis pipeline locally on your server or an HPC (High-Performance Computing) server and run it via the command line, or run it in our online Takara Bio Spatial Bioinformatics Portal.

You can follow the instructions to gain access on our Takara Bio Spatial Bioinformatics Portal.

Minimum system requirements:

• 256 GB RAM
• 64-core CPU

Compatible environments include:

• Single-server workstation
• HPC SLURM
• HPC SGE
• AWS