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.
- 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
If you are working with cell types that are small, or have low RNA content (small cells, nuclei), 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.
- 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