Question: Which tissue dissociation protocols are supported for use with the Fixed RNA Profiling assay?
Answer: Optimized tissue dissociation protocols for other single-cell assays can be used in the Fixed RNA Profiling Solution assay. Tissue dissociation protocols that have been previously tested for compatibility with the Single Cell Gene Expression assay (i.e., 3’ GEX) are expected to perform similarly in the Fixed RNA Profiling assay. If these isolation methods/protocols yielded good results for 3’ GEX, they are expected to perform similarly in FRP.
Fresh tissue can be dissociated into single-cell suspensions using the 10x Demonstrated Protocol: Tumor Dissociation for Single Cell RNA Sequencing. To thaw these cryopreserved samples, we recommend following the 10x Demonstrated Protocol: Thawing Dissociated Tumor Cells for Single Cell RNA Sequencing.
Alternatively, fresh tissue can be used in the Fixed RNA Profiling assay by following the 10x Demonstrated Protocol for Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling. This protocol may be preferred if you do not have an optimized tissue dissociation protocol for single cell suspensions.
Frozen tissue can be used in the Fixed RNA Profiling assay by following the 10x Demonstrated Protocol for Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling or by isolating nuclei using the Chromium Nuclei Isolation Kit.
The Tissue Fixation & Dissociation Demonstrated Protocol will be compatible with fresh and frozen tissue. It is expected to be released later in Q2 2022. The fixation of whole tissues for use in the Fixed RNA Profiling assay will not be supported until then. Tissue samples must first be chopped into smaller pieces before fixation for optimal assay performance. This step is required for uniform fixation and permeabilization.
We recommend performing pilot experiments to optimize sample preparation methods for specific tissue types before committing to large-scale studies.
Products: Fixed RNA Profiling Gene Expression