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 remains a significant technical challenge and usually requires customization for different tissue and tumor types. The workflow for nuclei isolation from your frozen sample consists of mechanical disaggregation of the tissue into a nuclear hypotonic isolation buffer without any enzymatic steps.
10x Genomics does not have any in-house protocols for nuclei isolation from frozen tissues, however 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.
- 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.
- 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
- 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.
- 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?
- 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'?
Products: Single Cell Gene Expression.