Why are nuclei samples more sensitive to RNase contamination than live-cell samples?

Answer:
During live-cell preparation, most cells remain intact and intracellular RNases are largely confined within the cytoplasm.

In contrast, nuclei isolation requires deliberate disruption of the cell membrane. This process releases cytoplasmic contents, including substantial amounts of endogenous RNases, into the suspension. Even after washing steps, residual RNases may remain associated with the nuclear membrane or persist in solution.

As a result, nuclei-based workflows are inherently more vulnerable to RNA degradation, especially for RNase-rich tissue types.

For this reason:

• Live-cell scRNA-seq is generally preferred whenever intact cells can be obtained for better RNA integrity
• Nuclei workflows are mainly recommended for frozen tissue or difficult-to-dissociate samples

ATAC + RNA multiome assays must use nuclei, because transposase access requires exposed chromatin.