Question: Can I generate custom probes to spike into the Visium Spatial Gene Expression for FFPE Transcriptome Probe Kit?
Answer: As instructed in our documentation, we recommend using the 10x Visium Spatial Gene Expression for FFPE Transcriptome Probe Panel. It has been extensively tested and validated with many tissue types (See: Visium Spatial Gene Expression for FFPE Tested Tissues List). It may be possible to spike-in custom probes into our Human or Mouse Transcriptome Probe Panels with that in mind.
Please note that custom probe spike-in approaches have not been tested or validated by 10x Genomics and are not supported. We cannot provide specific experimental guidelines and only provide the general recommendations in this article.
Resources:
- We recommend reviewing our Visium Spatial Gene Expression for FFPE: RNA-templated Ligation Probe Design Technical Note. This Technical Note on RNA-templated Ligation (RTL) provides general guidance on how candidate probes were identified and screened for performance.
- We suggest studying the Introduction of the Visium Spatial Gene Expression for FFPE User Guide. This section includes schematics of the workflow steps that can help in understanding the roles of each component of the probe pairs.
- We would next advise reviewing SciLife’s preprint Dual spatially resolved transcriptomics for SARS-CoV-2 host-pathogen colocalization studies in humans in which they utilized custom (SARS-CoV-2) probes during their Visium Spatial Gene Expression for FFPE experiments.
Considerations:
- As with primer design, the GC content and potential for off-targets should be considered carefully when designing probes. The RTL Probe Design Technical Note includes factors that influenced the decision to include or exclude certain probes from the Visium Spatial Gene Expression for FFPE Human or Mouse Transcriptome Probe Panels.
- Probe pairs used during Visium for FFPE require specific sequences. Effective capture of ligated probe pairs requires a synthetic poly(A) tail while Sample Index PCR (SI-PCR) requires molecules with Read 1 and Read 2S sequences for amplification. The RTL Probe Design Technical Note, Introduction of the Visium Spatial Gene Expression for FFPE User Guide, and Supplementary Table 12 of the SciLife preprint include information on these sequences.
- Concentration and purification of oligos, in addition to custom probe spike-in conditions, are in the Methods section and Supplementary Table 12 of the preprint.
Product: Visium for FFPE