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) or Nuclei Isolation from Mouse Brain Tissue for Single Cell ATAC Sequencing (CG000212) for preparing nuclei. These protocols outline how to isolate nuclei from both cell suspensions and frozen tissue.
The protocol has been validated on a variety of cells and tissues. With further optimization, these Demonstrated Protocols may be applicable to additional sample 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. The use of swing-bucket rotors has also improved final nuclei recovery in some instances.
- Removing background DNA: If background DNA is an issue, sorting cells prior to nuclei isolation may help remove background DNA. Treating the cells with DNase I prior to nuclei isolation can remove the ambient DNA, thus improving the quality of Single Cell ATAC libraries.
Product: Single Cell ATAC