I am preparing to have single cell RNA seq done on mouse tissue and I am using a protocol for single cell suspension we use for flow cytometry: Resect tissue, mince, digest in buffer, lyse red blood cells, Percoll gradient, wash.
I am deciding whether to include DNase enzyme in the digestion buffer along with collagenase. Most protocols that I can find for the single cell suspension do not include DNase. 10X Genomics also does not recommend using DNase because (obviously) if any of the active enzyme remains during the library generation, it will damage cDNA. However, I care about the immune cell population in my tissue, and I want to avoid promoting damage-associated molecular pattern (DAMP) signaling due to extracellular DNA, which may leak from cells that are damaged during the mince step. Will the subsequent re-suspensions and wash steps clear the enzyme sufficiently? Should I consider decreasing my working concentration? Has anyone examined this?