If you think your antibody didn’t work in CUT&Tag, the first thing to do is double check your sample prep and control reactions.
When troubleshooting CUT&Tag, it can be difficult to determine the exact cause. If a reaction fails, it could be a problem with the antibody, or it could be an issue with sample prep, the CUT&Tag workflow, or library amplification. For this reason, it is imperative to include positive (H3K4me3, H3K27me3) and negative (IgG) controls in your workflow, along with the recommended quality control checks for sample prep.
Begin by ensuring that you have a high-quality sample prep, are using the correct number of nuclei, and have a high percentage (>80%) of nuclei bound to ConA beads.
Examine CUT&Tag library yields for control reactions. H3K37me3 should have greater yields than IgG, while H3K4me3 may be slightly greater than or roughly equal to IgG.
Confirm that final purified CUT&Tag libraries have the expected size distribution on TapeStation/Bioanalyzer traces (~300 bp).
Analyze sequencing data for control reactions, including SNAP-CUTANA Spike-in controls. If the correct spike-in nucleosomes are recovered, and sequencing tracks show expected enrichment with low background, your workflow is validated.
If your experiment meets all these criteria, but reactions to experimental targets still fail to generate useful data, the issue is most likely your antibody.
In this case, we recommend sourcing additional antibodies to test. Source antibodies from multiple vendors. If CUT&Tag does not work for your target, consider using the CUTANA™ CUT&RUN Kit, which is robust for most targets and can go from cells to sequencing DNA in 3 days.