Question: How do you isolate nuclei from snap-frozen tissue for 3’ gene expression profiling?
Answer: Isolating nuclei from snap-frozen tissues for gene expression analyses has been a significant technical challenge and usually requires customization for different tissue and tumor types. 10x Genomics Recommends using the Chromium Nuclei Isolation kit for isolating nuclei from frozen tissues samples. The Chromium Nuclei Isolation Kit is an all-in-one sample preparation kit for generating high-quality nuclei suspensions from frozen tissue for use with many 10x Single Cell assays, including the 3' Single Cell Gene Expression assay. The Chromium Nuclei Isolation kit has broad applicability across tissue types which reduces requirements for sample preparation optimization or the need for tissue-specific protocols. The short nuclei isolation protocol with this kit reduces sample degradation and results in improved data quality.
- Work quickly and minimize handling steps. There is a balance between hands-on sample processing and minimal manipulation.
- Be sure to start with sufficient material. Nuclei loss is expected during the whole process. The Chromium Nuclei Isolation Kit is supported using 3-50mg of starting tissue.
General guidelines for nuclei isolation from snap-frozen tissue:
- After harvesting the tissue, the tissue should be washed in a petri-dish with cold PBS to remove blood and then using a rolled-up laboratory wipe, excess blood or solution should be absorbed from the surface of the tissue to limit ice crystal formation during freezing.
- Chop up the tissue into small pieces, the size of a rice grain for ease of freezing
- To flash freeze, either submerge the cryovial in liquid nitrogen, or a liquid-nitrogen cooled bath (e.g. isopentane), or place the tube deep in a bucket of dry ice. Wait at least 2-3 minutes for the tissue to freeze all the way through, and transfer the tube containing the tissue to vapor-phase liquid nitrogen for long-term storage.
- Tissues should be stored in a cryovial in liquid nitrogen for best results. Tissues can be stored short-term (1-2 days) at -80C if needed.
- Once tissues are removed from liquid nitrogen, the tissue should be kept at -80˚C on dry ice until use. All steps should be performed on ice or at 4˚C. Pre-chill all tools and reagents to 4˚C. The RNA in the tissue is stable while frozen at -80ºC or liquid nitrogen, but thawing the tissue prior to or during its dissociation can result in RNA degradation.
- In general, we do not recommend freezing nuclei as the nuclear membrane may be compromised during the freezing and thawing process. Please refer to the following article: Is it possible to freeze nuclei prior to Single Cell 3'?
Prior to the development of the Chromium Nuclei Isolation Kit, 10x Genomics did not have any in-house demonstrated protocols for nuclei isolation from frozen tissues. If not using the Chromium Nuclei Isolation Kit, there is a protocol for nuclei isolation from frozen tissues that some 10x customers have had success with. It is the "Frankenstein protocol" on the 10x Community site.
Note: Customer-Developed protocols are provided for general information only and are NOT directly supported, endorsed, or certified by 10x Genomics.
Below are some general guidelines that can be helpful when optimizing nuclei isolation from snap-frozen tissue.
Guidelines for the "Frankenstein protocol":
- Mechanically disaggregate the tissue into small pieces and then add to the cold nuclear isolation buffer (lysis buffer). We recommend using RNase inhibitors in the Lysis Buffer.
- If major clumps are seen in the sample, a gentler lysis condition can be tried by lowering the detergent concentration of the Lysis Buffer.
- A time-course should be performed for each sample type to determine the optimal lysis times to obtain high-quality nuclei. Read more: How can I run a lysis timeline to optimize nuclei isolation for 3' single-cell gene expression?
- For additional best practices for working with nuclei, see: What are the best practices for working with nuclei samples for 3' single-cell gene expression?
Products: Single Cell Gene Expression.