Question: Which nuclei isolation protocols are supported with the Fixed RNA Profiling assay?
Answer: The Fixed RNA Profiling assay has been validated with nuclei isolated using Demonstrated Protocol CG000124, our supported protocol for isolating nuclei from cells or fresh tissues using the Chromium Single Cell 3' Gene Expression assay, or the Chromium Nuclei Isolation Kit.
Nuclei 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 the nuclei isolation method/protocols yielded good results for 3’ GEX, nuclei are expected to perform similarly in FRP.
Nuclei purification methods are also compatible with the Fixed RNA Profiling assay Nuclei samples can be FACS sorted to remove subcellular debris, aggregates/clumps, and ambient RNA/DNA. The sorted single-nuclei solution should be used immediately as input to Sample Fixation. Extra care should be taken to avoid nuclei damage as sorting can be stressful and compromise the nuclear membranes, leading to leakage of nuclear content. Density gradients using Iodixanol (OptiPrep™) or a modified sucrose gradient can also be used for cleaning up a nuclei prep. Additional information can be found in What are the best practices for working with nuclei samples for 3' single-cell gene expression? Although this article is relevant for 3' GEX, the recommendations for Fixed RNA Profiling are the same.
Nuclei can be isolated from frozen tissue using the Chromium Nuclei Isolation Kit. Isolating nuclei from frozen tissues can be technically challenging and may require customization for different tissue and tumor types. We recommend performing pilot experiments to access nuclei data quality and optimal sample preparation methods for tissue type of interest before committing to large-scale studies.
While working with nuclei samples, it is critical to have RNAse inhibitors in the lysis, wash, and resuspension buffers. Similar to the 3'GEX assay, we recommend a concentration of 0.2U/ul of RNAse inhibitor (Protector RNase inhibitor: Sigma, PN-3335399001).
Clumping is an important consideration for the use of nuclei in the Fixed RNA Profiling assay. To minimize effects on downstream assay performance, we recommend filtering nuclei samples with 30 µm filters (Miltenyi Pre-Separation Filters or Sysmex CellTrics Filters) post-fixation to help reduce nuclei clumping/aggregates.
Please note: A minimum of 500,000 nuclei per sample is required for sample fixation in the Fixed RNA Profiling assay.