Question: How do I dissociate my tissue of interest?
Answer: We have not tested dissociating all tissue/sample types. General guidelines for preparing optimal single-cell suspensions can be found in the Cell Preparation Guide (https://support.10xgenomics.com/single-cell-gene-expression/index/doc/demonstrated-protocol-single-cell-protocols-cell-preparation-guide).
The following articles may also be helpful :
- What are the best practices for dissociating tissue samples into single cells?
- Should I use warm or cold tissue dissociation?
Below are some resources that may be helpful when considering methods for dissociating your tissue of interest.
1. Demonstrated Protocols
Dissociation of Mouse Embryonic Neural Tissue for Single Cell RNA Sequencing (https://support.10xgenomics.com/single-cell-gene-expression/index/doc/demonstrated-protocol-dissociation-of-mouse-embryonic-neural-tissue-for-single-cell-rna-sequencing)
Although written for mouse embryonic neural tissue, this protocol provides a good overview of how to prepare fresh tissue for the Single Cell 3' assay. The specific enzymes and incubation times may be different depending on tissue type and will need to be optimized. Generally, the cell viability should be > 80% and between ~200 and 18,000 cells should be loaded per sample. If this is the first time a specific cell/tissue type is being run, it is generally recommended to target ~2,000 cells.
Tumor Dissociation for Single Cell RNA Sequencing (https://support.10xgenomics.com/single-cell-gene-expression/sample-prep/doc/demonstrated-protocol-tumor-dissociation-for-single-cell-rna-sequencing)
This protocol outlines best practices for freshly obtained (not frozen) tissue dissociation of mouse melanoma, colon tumor, and breast tumor for use in 10x Genomics Single Cell protocols.
Modifications to this demonstrated protocol may be necessary for other tumor types (e.g. dissociation time, resuspension buffer, enzyme concentration, centrifugation speed, and time).
2. Commercially available solutions for tissue dissociation
Several companies have products designed specifically for tissue dissociation; we recommend consulting their websites for information: i) Miltenyi Biotec, ii) Worthington Biochemical, iii) Sigma-Aldrich, and iv) Roche Diagnostics. Some of the products have been validated in-house (e.g. Miltenyi Micro Beads).
3. Published dissociation methods
We recommend the Worthington Biochemical website for recommendations and published protocols on specific tissue types: http://www.worthington-biochem.com/tissuedissociation/default.html
It may also be useful to see methods other researchers have used for a specific tissue type. A list of 10x Publications that is searchable by assay, species, and tissue type is available here: https://www.10xgenomics.com/resources/publications
4. Customer developed protocols
Customer developed protocols for tissue dissociation are available here: https://www.10xgenomics.com/resources/customer-developed-protocols
Note: Customer-Developed protocols are provided for general information only and are NOT directly supported, endorsed, or certified by 10x Genomics.
Products: Single Cell Gene Expression, Single Cell Immune Profiling, Single Cell ATAC