Question: Can I sort nuclei for Single Cell ATAC sequencing or Single Cell Multiome ATAC + GEX?
Answer: When sorting nuclei to be used for single-cell ATAC or single-cell Multiome ATAC+GEX assays, care should be exercised as sorting and additional handling of nuclei can further compromise the nuclear membrane, leading to leakage of nuclear content. Please read this article: What are the best practices for flow sorting cells for 10x Genomics assays? Nuclei sorting is not recommended if the user cannot retrieve at least 500,000 nuclei post-sorting. Additionally, certain dyes used for nuclei sorting, intercalate between the DNA and disrupt chromatin structure. The choice of sorting dye is, therefore, an important consideration.
In the plot below, nuclei were stained with propidium iodide (PI), an intercalating dye, and sorted based on the population staining positive for PI. After sorting, the sample had a reduced number of unique fragments per barcode, reduced targeting, and lower clustering resolution within the tSNE plot.
Other sorting dyes tested shown to impact data quality: DAPI, Ethidium Homodimer, Vybrant Dyecycle Green, Zombie Violet.
Alternatives: 7-Aminoactinomycin D (7-AAD) has shown reproducible success for both ATAC and Multiome ATAC+GEX assays. This dye maintains high sensitivity and targeting, while also reducing noncell/background reads.
Sorting guidelines can be found in the following demonstrated protocol. Some key points highlighted in this protocol are that nuclei should not be sorted after permeabilization. The sort collection buffer should contain sufficient RNAse inhibitor to account for dilution during sorting.
Products: Single Cell ATAC, Single Cell Multiome ATAC + GEX