Question: Can I perform both Single Cell Gene Expression and Visium Spatial Gene Expression experiments on my OCT-embedded tissue block?
Answer: If performing both Spatial and Single Cell Gene Expression on the same fresh frozen OCT-embedded tissue block, we would recommend acquiring good sections for the Visium assay, before collecting sections for the Single Cell Gene Expression assay, as tissue collection for nuclei isolation may lead to exhaustion of the tissue block for smaller samples.
Single Cell Gene Expression (i.e., 3' assay)
We have done limited testing using OCT-embedded tissue for Single Cell Gene Expression (3' assay) experiments.
- Using the Chromium Nuclei Isolation Kit:
The Chromium Nuclei Isolation Kit with fresh frozen OCT-embedded tissue is not fully supported by 10x Genomics. However, limited testing with OCT-embedded tissue (10-100µm mouse brain curls) showed positive results. Please note that tissue quality may decrease due to extra sample processing of OCT embedded tissues.
Best Practices when isolating nuclei from OCT-embedded tissues:
- Run a pilot study, when possible, to determine whether a particular OCT-embedded tissue is compatible with the Chromium Nuclei Isolation Kit. Measuring the RIN score of the tissue block is recommended when the block quality is unknown or compromised.
- To maintain RNA integrity, minimize freeze-thaw and sectioning cycles before nuclei isolation from OCT curls.
- For adequate nuclei yield, we recommend starting with curls with a thickness of >40µm.
- To reduce the risk of clogs, remove as much excess OCT from the tissue block before isolating nuclei.
- When working with OCT-embedded tissues, remaining OCT can cause GEMs to appear cloudy. This is normal and expected.
For more information, please see:
- Using the Customer-Developed Frankenstein Protocol:
In those tests, 3-4x 100µm sections were collected from fresh frozen OCT-embedded mouse brain and human breast cancer tissue blocks. OCT was physically removed from the tissue using cold forceps, followed by nuclei isolation using the customer-developed Frankenstein protocol (protocol found on our webpage of Community Developed Protocols).
Single Cell and Visium Gene Expression data can be combined using 3rd party tools to elucidate spatiality in single cell data and improve resolution in Visium data. Please refer to the Analysis Guides for more details.
Note: 10x Genomics does not provide support for community-developed protocols and tools and makes no guarantees regarding their function or performance.
Single Cell Gene Expression Flex (i.e., Fixed RNA Profiling)
Based on limited testing, we have successfully generated Single Cell Gene Expression Flex (i.e., Fixed RNA Profiling) libraries from OCT embedded fresh frozen tissue. For guidance using OCT embedded fresh frozen tissue with the Flex/Fixed RNA Profiling assay, please review this article: Can I use OCT embedded fresh frozen tissue with the Flex/Fixed RNA Profiling assay? The modified version of the Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling Demonstrated Protocol outlined linked in this article is the preferred sample preparation method for OCT embedded fresh frozen tissue for use in the Flex/Fixed RNA Profiling assay. If the Chromium Nuclei Isolation Kit is used, significantly more sections/curls will be required due to variability in nuclei yield.
Please note: This modified protocol in this linked article has only been tested in a limited capacity for OCT embedded fresh frozen tissue. A pilot experiment is recommended for your specific tissue type of interest prior to committing to a larger study. We have not tested OCT embedded fixed frozen tissue with or without sucrose cryoprotection. If using OCT embedded fixed frozen tissue, we recommend proceeding directly to tissue dissociation. Please note that this guidance has not been tested for OCT embedded fixed frozen tissue, and a pilot experiment is recommended.
Product: Visium for Fresh Frozen, Single Cell Gene Expression, Single Cell Gene Expression Flex, Fixed RNA Profiling