Can I do qPCR or Bioanalyzer/TapeStation to evaluate raw CUT&RUN DNA?
TapeStation/Bioanalyzer and qPCR 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 as to why.
Why can’t I use qPCR?
- qPCR is used in ChIP to verify the enrichment of a known on-target region compared to the bulk chromatin input. However, CUT&RUN is performed in intact cells. There is no immunoprecipitation step and no bulk chromatin input for comparison. Simply stated, there is no way to properly control or analyze qPCR.
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.