Question: How much residual membrane is acceptable when preparing nuclei for the Single Cell ATAC or Single Cell Multiome ATAC + Gene Expression assays?
Answer: Residual membrane is acceptable on nuclei prepared for the Single Cell ATAC and Multiome assays. Although removing excess membrane is preferred, customers have reported that when conducting lysis timelines for some cell types, removing the residual membrane cannot be achieved without degrading nuclei quality. In these cases, residual membrane is preferred to nuclei overlysis and high quality data could be still generated.
If you may have nuclei with residual material, carefully assess:
- Whether the material is residual membrane/cytoplasm (acceptable) or leaking material from damaged nuclei (should be minimized). These are best distinguished using high magnification brightfield microscopy; low magnification is frequently insufficient to confidently assess nuclei quality (see How can I assess the quality of my nuclei for Single Cell ATAC or Single Cell Multiome ATAC+GEX Sequencing?).
- Whether the sample contains viable cells or nuclei with residual membrane. Viable, intact cells generate minimal ATAC data since the transposase cannot access chromatin. Cells that stain dead with a viability dye have ruptured plasma membranes and have chromatin that is accessible to the transposase. A fluorescent viability dye such as AO/PI or Ethidium homodimer-1 is highly recommended to confirm that nuclei with residual membrane have achieved our target of <5% viable cells after nuclei isolation.
Products: Single Cell Multiome ATAC+GEX, Single Cell ATAC