Question: Are there considerations for small cells or cells with low RNA??
Answer: Small cells may need additional considerations as they tend to contain less RNA than larger cells. Likewise, larger cells with low RNA content and nuclei samples may need additional considerations.
Additional Considerations:
- Extensive manipulation used during sample preparation (dissociation, FACS, any conditions that cause lysis, etc.)
- Cell viability (low cell viability can reduce transcriptional output as cells enter apoptosis)
- PCR cycling conditions
Recommendations:
If you are working with cell types that are small, or have low RNA content (small cells, nuclei), and consistently experiencing low cDNA yields despite final quality that meets expectations, experiment with adding an extra 1-2 PCR cycles in the cDNA amplification and/or Sample Index PCR steps. However, keep in mind that the extra PCR cycles may increase PCR artifacts and may not increase final library complexity.
Notes:
- Primary cells and cells of small cell size (i.e. PBMCs) generally have lower RNA content compared to immortalized cell lines or cancer cells.
- We have successfully generated libraries from cDNA yields as low as ~1-2 ng.
- A typical cDNA yield of 1000 RNA-poor cells is <5 ng/ul
- A typical cDNA yield of 1000 RNA-rich cells is >10 ng/ul
Products: Single Cell Gene Expression / Single Cell Immune Profiling