Question: Can I add enzymes (such as DNase) or other chemical additives to my cell suspension?
Answer: The use of collagenase, dispase, accutase, DNase I, or TurboNuclease during sample preparation in the Single Cell 3' or 5' assay have not been tested. However, the following information may be helpful:
Collagenases, Dispase, Accutase: A number of customers have used these enzymes for tissue/cell dissociation successfully in sample preps with our Single Cell assay.
Nucleases: If DNases are not washed away and retain residual activity after GEM-RT Incubation, they will degrade the cDNA.
Chemical additives in buffers, such as Mg2+ and EDTA, will interfere with the RT reaction and inhibit the RT enzyme. We have not observed reduced assay performance with Ca2+.
In general, we recommend washing the cells at least once in 1X PBS + 0.04% BSA prior adding to adding the cells to the RT reaction. Additional washes can be performed to remove possible contaminants and dead cells. Ideally, the cell stock concentration will be high enough (700-1200 cells/ul) prior to adding the cells to the RT reaction Master Mix. To further reduce the risk of contaminant carryover, the total volume of cell suspension should be minimized that is added to the Master Mix.
Products: Single Cell 3', VDJ