Should digitonin be included in my wash buffers for my entire CUT&Tag experiment?

Yes, digitonin needs to be present in all wash buffers during the entire CUT&Tag experiment. This is because cell membrane permeabilization caused by digitonin is reversible. Removing or lowering the amount of digitonin in the buffers may interfere with the ability of the antibodies and pAG-Tn5 enzyme to enter the nuclei.

Cells are more sensitive to the concentration of digitonin than to the digitonin treatment time. If you find that the recommended amount of digitonin is leading to extensive cell lysis, you can optimize the concentration of digitonin in your assay as described in the CUT&Tag Assay Kit Protocol, Appendix C: Determination of Cell Sensitivity to Digitonin.

Last updated: April 30, 2024