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 cell pellet 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.
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.
Additional steps can be taken to improve cell/nuclei yield. Please review: How can I improve my cell/nuclei recovery using the Fixed RNA Profiling Assay?
Products: Fixed RNA Profiling Gene Expression, Single Cell Gene Expression Flex