Assay

CUT&RUN and CUT&Tag: sanity checks and pitfalls

Protein A-MNase (CUT&RUN) or protein A-Tn5 (CUT&Tag) is tethered to the antibody in situ, so background is far lower than ChIP-seq and libraries need only a few million reads. That low background breaks assumptions built into ChIP-seq tools: MACS2 with default settings and an IgG control behaves differently, spike-in normalization (E. coli DNA carried by the enzyme) is the honest way to compare samples, and fragment-size classes carry information about what was cut. Paired-end sequencing and fragment-level analysis are not optional.

Who this is for: Labs that replaced ChIP-seq with CUT&RUN or CUT&Tag for low cell numbers and are surprised that ChIP-seq peak-calling defaults over-call or under-call on the much cleaner, much sparser signal.