Question: How can I optimize my nuclei prep for Single Cell ATAC sequencing?
Answer: A high-quality nuclei suspension is critical for Single Cell ATAC sequencing. We recommend starting with the Nuclei Isolation for Single Cell ATAC Sequencing Demonstrated Protocol (CG000169) for preparing nuclei. This protocol outlines how to isolate nuclei from both fresh and cryopreserved cell lines, and as well as more fragile primary cells. As many samples may have limiting sources and material, we have also provided guidance for low input samples.
The protocol has been validated on PBMCs and cells lines. With further optimization, the Demonstrated Protocol may be applicable to most cell types. Below are steps that may need to be optimized:
- Lysis time: Optimal lysis times to obtain high-quality nuclei can be determined by performing a lysis time course. Testing different lysis times on your sample until obtaining <5% live cells will help determine the optimal lysis time. For robust sample types, a broader timeline (0, 5, 10 minutes) is a good starting point. For delicate sample types, finer timepoints may be more appropriate (0, 2, 3, 4, 5 minutes). Avoid overlysing the cells, which will compromise nuclear membrane integrity. Refer to the article "How can I assess the quality of my nuclei for Single Cell ATAC Sequencing?" for guidance on counting and visualization.
- Lysis Buffer: We recommend using our lysis buffer first as it has been optimized to work with the assay and to achieve minimal mtDNA contamination. Alternative lysis buffers may be required for unique sample types but may impact assay performance.
- Centrifugation: In cases where low nuclei recovery is an issue, centrifugation time may be increased in an attempt to improve recovery.
- Removing background DNA: If background DNA is an issue, sorting cells prior to nuclei isolation may help remove background DNA. We do not recommend sorting nuclei, as sorting may damage the cellular membrane and decrease assay performance.
Product: Single Cell ATAC