What positive and negative controls can I use to demonstrate that my CUT&Tag experiment is successful?

We recommend using a CUT&Tag-validated positive control antibody, like our Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751, in your experiment to confirm that your CUT&Tag assay is working regardless of the performance of your desired target-specific antibody.

For the negative control, either the IgG antibody or the input DNA sample can be used, depending on the requirements of the peak calling algorithm used. Both negative control options have their own advantages and limitations.

• Input DNA as a negative control: The input DNA sample is fragmented genomic DNA that is not tagmented by Tn5, so it requires additional benchwork compared to the CUT&Tag samples from the same experiment, including sonication fragmentation and in-vitro adaptor ligation steps for library preparation. The input DNA sample will always generate enough library yield and sequencing reads for data analysis. CUT&Tag signals are so strong, however, that some background signal can be counted as peaks if you are using the input DNA sample as the negative control.
• IgG antibody as a negative control: The issue described above can be avoided by using an IgG antibody as a negative control, as it performs better with regard to reasonable and reliable peak calling results. However, using an IgG control runs the risk of obtaining a lower library yield with significantly fewer sequencing reads for your data analysis.

Note that some labs analyze CUT&Tag peaks without any negative controls.

The following control antibodies are included in our CUT&Tag Assay Kit #77552: Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751, Normal Rabbit IgG #2729, and Normal Mouse IgG #68860.

Last updated: September 12, 2024