This article also applies to meCUT&RUN and Multiomic CUT&RUN.
We do not recommend using TapeStation/Bioanalyzer or qPCR to evaluate raw CUT&RUN DNA, as they will NOT provide useful information at this step of the workflow. CUT&RUN yields are too low for these methods. Read on for more information or see this blog.
Why can’t I use qPCR?
It is generally not recommended to perform qPCR directly on raw CUT&RUN DNA prior to library preparation. CUT&RUN yields are typically very low—often in the sub-nanogram range—and the fragments have not yet undergone end repair or adaptor ligation. At this stage, DNA ends are heterogeneous and amplification efficiency can be inconsistent, which makes qPCR results unreliable and difficult to interpret. In addition, using material for qPCR consumes precious template that is better preserved for library construction. For these reasons, enrichment and specificity are more accurately assessed after library preparation, when adaptor-ligated libraries provide sufficient and uniform template for reliable quantification and quality check prior to sequencing.
Why can’t I use the Bioanalyzer/TapeStation?
CUT&RUN uses intact cells bound to a solid support and selectively cleaves antibody-bound chromatin. These advances bypass bulk chromatin fragmentation and immunoprecipitation, resulting in high signal-to-noise and low cell input requirements vs ChIP. As a result, however, raw CUT&RUN DNA yields are often below the limit of sensitivity for fragment size distribution using the TapeStation or Bioanalyzer.