As in any experiment, be it OMICS or anything else, a control has to come from the exact same day/run/batch. It has to be done alongside the IP, you cannot just randomly pull controls from somewhere.

Hello,

Ideally, yes, but with pAG-MNase it is not strictly required.

In CUT&RUN, the IgG control serves to establish the nonspecific background cleavage profile. For this background to accurately reflect what your target antibody would produce in the absence of specific binding, the IgG control should be:

• Same species as your target primary antibody — so that pAG-MNase binds both with equivalent efficiency.

• Same subclass where possible — Protein A and Protein G have different affinities across IgG subclasses (e.g., mouse IgG1 binds weakly to Protein A).

The 2019 engineered pAG-MNase (Meers et al.) greatly improved cross-species binding, so a non-matched IgG will still be recruited to chromatin. However, binding efficiencies still differ between species/subclasses — rabbit IgG binds more tightly than mouse IgG1, for example. If the IgG control is not species-matched, its background cleavage pattern may not faithfully reflect your target antibody's background, reducing peak-calling accuracy.

Recommendation: Use a normal IgG from the same host species and subclass as your target antibody. If that's not available:

• Ensure the IgG control is from a species that pAG-MNase binds efficiently (rabbit, mouse IgG2a/2b/3, rat, goat all work well), and note this limitation in your methods.

• For mouse IgG1 primaries, adding a rabbit anti-mouse secondary antibody is advised to boost pAG-MNase recruitment.

Best regards, Alisa Liu