Question: Can post-fixation samples be sorted in the Fixed RNA Profiling assay?
Answer: Post-fixation samples can be flow sorted for advanced sample clean-up to remove cellular debris from single-cell or nuclei suspensions using 7-AAD. It is always recommended to consult a core facility or an experienced flow user before initiating a flow sorting experiment.
Best practices for sorting fixed samples:
- Samples should be collected in PBS + 1% BSA supplemented with RNase inhibitor (Protector RNase inhibitor from Sigma, PN-3335399001). We recommend a final concentration of 0.2 U/ul of RNAse inhibitor.
- Samples should be spun down and resuspended in PBS + 0.02% BSA before proceeding with probe hybridization.
We have not tested the enrichment of specific populations post-fixation in the Fixed RNA Profiling Assay. Sample fixation may change the presentation of the epitopes on the surface of the cells. Additional optimization may be required for sorting samples post-fixation. We hope to develop additional guidance in the future but currently do not have a timeframe.
Samples can be enriched for labeled cells using FACS to identify specific populations of interest, including rare subpopulations, before sample fixation. Cells should be labeled following the Cell Surface Protein Labeling for Chromium Fixed RNA Profiling for Singleplexed Samples with Feature Barcode technology. Guidance for FACS can be found in the Appendix of this Demonstrated Protocol. Sorted cells should be counted and viability measured before proceeding to the Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling Demonstrated Protocol.
Products: Fixed RNA Profiling Gene Expression