Question: How do I perform a lysis timeline to optimize my nuclei isolation for Single Cell ATAC sequencing?
Answer: Optimizing cell lysis is a key step for isolating quality nuclei for Single Cell ATAC sequencing. Refer to our demonstrated protocols for specific reagent compositions, materials and handling steps.
Nuclei Isolation for Single Cell ATAC Sequencing (for cell lines and fragile cell suspensions):
Nuclei Isolation from Mouse Brain Tissue for Single Cell ATAC Sequencing:
Each of the demonstrated protocols contains a cell lysis step, in which the tissue or cell pellet is incubated in the presence of Lysis Buffer. The Lysis Buffer contains 3 detergents that will solubilize the cellular membrane and permeabilize the nuclear membrane. This allows for the transposition reagents to enter the nucleus and transpose the adaptor sequences into the genome. Optimizing the amount of time the sample spends in the Lysis Buffer is critical to ensuring that the majority of the cell membranes are lysed so that the transposition reagents can access the nucleus, while preventing over-lysis of the nuclear membrane, which can result in leakage of nuclear contents and subsequent increase in background signal.
If using the Chromium Nuclei Isolation Kit, please review the following article: Do I need to optimize any lysis conditions when using the Chromium Nuclei Isolation Kit?
Below you will find some guidelines to go about 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 robust cell types, a broader timeline may be appropriate (i.e. unlysed, 5 min, 10 min, 15 min, 20 min...)
- For sensitive cell types, a narrower timeline may be appropriate (i.e. unlysed, 2min, 3 min, 4 min, 5 min...)
2. Determine the appropriate Lysis Buffer concentration.
- For cell lines and cells already in suspension (PBMCs), a 1x Lysis Buffer is recommended as a starting point.
- For tissues, a 0.1x Lysis Buffer is recommended as a starting point.
Refer to the respective demonstrated protocols linked above for buffer recipes. Please note that the 0.1x Lysis Buffer should be diluted with our Dilution buffer (recipe in the protocol) and not water.
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 as indicated in the demonstrated protocol
- After the completion of each timepoint, add wash buffer and spin down immediately as indicated in the demonstrated protocol
- Remove the supernatant and resuspend in diluted Nuclei Buffer as indicated in the demonstrated protocol
- Using trypan blue or another live/dead stain and proceed to count the sample as indicated in the demonstrated protocol
- 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 as indicated on the demonstrated protocol and incubate on ice
- After reaching your first timepoint, remove a small aliquot (~5-10uls), add wash buffer, and spin down. Resuspend in a small volume of diluted nuclei buffer (~10uls).
- Using trypan blue or another live/dead stain, proceed to count the sample as indicated in the demonstrated protocol.
- 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.
4. Determine optimal lysis time
Optimal lysis time is reached when the sample stains >95% dead (<5% live), while still showing quality nuclei as shown in the following Q&A article: How can I assess the quality of my nuclei for Single Cell ATAC Sequencing?
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