Question: How can I run a lysis timeline to optimize nuclei isolation for 3' single-cell gene expression?
Answer: Having a high-quality nuclei sample is one of the most important prerequisites for a successful experiment. Optimizing the amount of time the sample spends in the Lysis Buffer is critical to ensuring that most of the cell membranes are lysed while preventing the over-lysis of the nuclear membrane, which can result in leakage of nuclear contents and subsequent increase in background signal. Below you will find some general guidelines for performing your own lysis timeline for your specific sample type.
1. Determine the number of time points and lysis times you are going to try.
- For single-cell suspensions and sensitive tissue types, a narrower timeline may be appropriate (i.e., unlysed, 1 min, 2 min, 3 min, 4 min ...)
- For robust tissue types, a broader timeline may be appropriate (i.e., unlysed, 2 min, 5 min, 10 min, 15 min ...)
2. Determine the appropriate Lysis Buffer concentration.
- For cell lines and cells already in suspension (PBMCs), we recommend 0.025% NP40 as a starting point.
- For fresh mouse brain tissue, our in-house experiments were performed with 0.1% NP40. Alternative concentrations may be appropriate for other tissue types.
3. Carry out your timeline
For fresh tissue and cell lines:
- Using a non-precious sample, split your cell suspension (from cell lines or dissociated tissue) into multiple aliquots, each containing the same number of cells.
- Spin each of the aliquots down and carefully remove the supernatant
- Resuspend each of the aliquots (except for your unlysed control) in the Lysis Buffer
- After the completion of each timepoint, add wash buffer and spin down immediately.
- Remove the supernatant and resuspend in the final resuspension buffer.
- Using trypan blue or another live/dead stain and proceed to count the sample
- Record the number of live and dead cells, record observations of nuclei clumps, and observe the nuclei under the microscope (at least 40x magnification) to evaluate nuclei quality. Take images when possible.
- Repeat with additional timepoints
For frozen tissue*:
- Add Lysis Buffer to your sample--start timing!
- Dounce the tissue and incubate on ice.
- After reaching your first-time point, remove a small aliquot (~5-10uls), add wash buffer, and spin down. Resuspend in a small volume of final resuspension buffer (~10uls).
- Using trypan blue or another live/dead stain, proceed to count the sample.
- Record the number of live and dead cells, record observations of nuclei clumps, and observe the nuclei under the microscope (at least 40x magnification) to evaluate nuclei quality
- Repeat with additional time points.
* 10x Genomics does not have any officially supported protocols for isolating nuclei from frozen tissues for use with the 3' Gene Expression assay. See: How do you isolate nuclei from snap-frozen tissue for 3’ gene expression profiling?
4. Determine optimal lysis time
Optimal lysis time is reached when the sample stains >95% dead (<5% live) while still showing good quality nuclei, as shown in the following Q&A article: What are the best practices for working with nuclei samples for 3' single-cell gene expression?
Products: Single Cell Gene Expression, CellPlex