Question: Can I use fewer than the minimum recommended number of cells/nuclei during sample fixation or probe hybridization with the Fixed RNA Profiling Assay (i.e., Single Cell Gene Expression Flex assay)?
Answer: As some cell loss is expected throughout the assay, we recommend starting with recommended minimum number of cells/nuclei to ensure that there are sufficient cells/nuclei for the downstream workflow.
- For sample fixation, the recommended minimum of fresh cells or nuclei suspensions are 300,000 cells or 500,000 nuclei, respectively using the Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling Demonstrated Protocol.
- Singleplex: The recommended minimum input is 200,000 cells or 400,000 nuclei per hybridization. In addition, the recommended minimum input for FFPE dissociated suspension is 400,000 cells per hybridization.
- Multiplex: The recommended minimum input is 50,000 cells or 100,000 nuclei per hybridization (i.e., per probe barcode). In addition, the recommended minimum input for FFPE dissociated suspension is 100,000 cells per hybridization (i.e., per probe barcode).
- We recommend starting with 1 x 10^6 cells whenever possible, with a maximum input of 2 x 10^6 cells/nuclei per hybridization.
The recommended minimum number of cells/nuclei was set to ensure assay success as recovery can vary greatly between sample types. For fixation and hybridization, it may be possible to use less than the recommended minimum. However, there is potential risk of not having enough cells/nuclei for the downstream assay. The recommended minimum is not a strict requirement for assay performance, but this guidance ensures that there are enough cells/nuclei to proceed with the workflow.
Careful considerations, if the number of cells/nuclei are reduced below the minimum recommended number:
- There is potential risk of not having enough cells/nuclei for the downstream assay.
- Debris in sample post dissociation may cause counting inaccuracies, especially when using Trypan. This could result in using less than expected number of cells during hybridization.
Loss of cells/nuclei during fix, quench, and post-hyb washes.
- Losses varies by experience level and sample type.
- Loss is typically higher in fix, quench spins than post-hyb.
- Follow best practices to reduce cell/nuclei loss throughout the workflow outlined in this article: How can I improve my cell/nuclei recovery using the Fixed RNA Profiling Assay?
Amount of supernatant left behind during post-hyb washes may result in increase in wasted data:
- Drop in Reads Mapped to Probe Set
- Drop in Reads Mapped Confidently to Filtered Probe Set
- Drop in Valid Barcodes (multiplexing context)
More likely to observe atypical library trace, due to unligated LHS probe.
- If the Flex library looks atypical, please review this article: Why does my Fixed RNA Profiling library look unexpected or have low yield?
- In a multiplexing context, low starting cell input numbers may make it difficult to pool samples in equal number (ie drop outs may be more likely). When faced with a complicated pooling scenario, refer to pooling workbook for alternative pooling strategies (Link to workbook can be found on the User Guide landing page: Chromium Fixed RNA Profiling Reagent Kits for Multiplexed Samples) Pooling workbook has additional guidance to help evaluate tradeoffs for strategies that compromise on equal representation within a multiplexing pool in exchange for maximizing pellet size.
Products: Fixed RNA Profiling Gene Expression, Single Cell Gene Expression Flex