Is it possible to perform CUT&RUN on fixed frozen cells?

Yes. Cell fixation is recommended when the cells are very fragile (easily lysed during experiments), when the cellular signaling pathway is sensitive to the Conconavalin A beads used for cell binding, when the target protein binds weakly to DNA, or when you hope to freeze the cell pellets for future use. A light fixation (0.1% formaldehyde for 2 min) is critical for the success of CUT&RUN with fixed cells. A reverse crosslinking step is also required at the end of the assay to remove protein from DNA. We find that live cells generally have better performance than fixed cells in the CUT&RUN assay, therefore, we recommend using live cells if possible, unless fixation is necessary as noted in the above-mentioned scenarios.

Last updated: November 1, 2023