Ideally, the integrity and purity of RNA in a tissue sample should be established prior to nuclei isolation. While intact nuclei can be isolated from many tissues, in some instances the RNA associated with those nuclei will be too degraded and thus not suitable for subsequent analyses. Of particular concern, if RNA obtained from frozen tissue is low quality, successful single-nucleus molecular profiling is unlikely.
Although there are now many published protocols to isolate nuclei for RNA sequencing, most do not incorporate the specific quality control steps needed to ascertain RNA quality prior to enrichment. A new two-step procedure, described in “Isolation of nuclei from mammalian cells and tissues for single-nucleus molecular profiling” ((see Nadelmann, E. R., Gorham, J. M., Reichart, D., Delaughter, D. M., Wakimoto, H., Lindberg, E. L., Litviˇnukova, M., Maatz, H., Curran, J. J., Ischiu Gutierrez, D., Hubner, N., Seidman, C. E., & Seidman, J. G. (2021). Isolation of nuclei from mammalian cells and tissues for single-nucleus molecular profiling. Current Protocols, 1, e132. https://doi.org/10.1002/cpz1.132) reports an enhancement to most current enrichment procedures insofar as this all-important quality control step.
The tissue quality check in the new protocol uses RNA isolated from the same sample used to isolate the nuclei. The sample used for RNA isolation is evaluated for its purity and integrity. Assuming a pass, only then is the nuclei isolation procedure deployed. The disclosed protocols help prevent situations where that “perfect” nuclei is purified, only to find out the RNA is too degraded to be of value, making the downstream data worthless.
Overall, the new approach to nuclei isolation provides an efficient and uniform method of tissue dissociation, eliminates risk of contaminant exposure, reduces reagent requirements, and generally increases the efficiency of the purification process. The RNA quality control checks documented in the protocol effectively reduces the overall costs of downstream snRNAseq experiments. Finally, the quality of the nuclei isolated with this new protocol was confirmed to be good enough for downstream single cell sequencing and analysis.