Questions: How can I test the quality of my nuclei prep?
Answer: Having a high quality sample is one of the most important prerequisites for a successful experiment. It is difficult to determine the quality of a nuclei prep prior to sequencing, but here are some important considerations:
- Depending on the sample type, additional optimization of the 10x Demonstrated Protocol "Isolation of Nuclei for Single Cell RNA Sequencing" may be required. Specifically:
- Lysis conditions. Isolation of nuclei from cells requires disrupting the structural integrity of the cellular membrane while the nuclear membrane remains intact. Detergent-based lysis solubilizes the hydrophobic membrane proteins which releases the cell’s cytoplasmic contents (i.e. organelles, cytoplasmic nucleic acids, ambient RNA, proteases) without impacting the integrity of the subcellular components. Lysis efficacy should be assessed via microscopy after 3 – 5 min for single cell suspensions or ~15 min for neural tissue (a low fraction of viable cells <5% should be visible to ensure complete lysis). It is critical to avoid over- or under-lysing the cells. Cell lysis should be carried out on ice and using chilled reagents.
- Centrifugation rotor/speed/time, number of wash steps, and methods of debris removal (e.g. filtration, density gradient centrifugation [such as sucrose or Optiprep], myelin removal, or flow cytometry). For example, solid tissues and cryopreserved samples may require additional preparation steps to remove debris and minimize background mRNA contamination. Use of a swing-bucket (as opposed to fixed-angle) rotor during centrifugation steps may also increase recovery.
- In addition, nuclei should be visualized using microscopy and viability staining (note, nuclei will appear "dead" when staining with trypan blue). Inspection of nuclei will also allow you to visualize the level of debris and the degree of clumping. If your nuclei appear to be clumping into aggregates, it is possible to increase the BSA in the media to prevent this (2%).
If running nuclei for the first time, we would recommend also running a positive control, such as a cultured cell line with high viability (>90%) and yielding a sufficient number of nuclei (i.e. >1 million nuclei).
Products: Single Cell Gene Expression