What do adapter dimers look like in CUT&RUN and how do I avoid them?

Adapter dimers are generated by self-ligation of sequencing adapters that are preferentially amplified due to their small size. Adapter dimer contamination appears as a peak at ~125 bp (below), and is caused by low input DNA, inefficent adapter ligation, and/or using excess beads during library purification. Adapter dimers should represent no more than 5% of the total 200-700 bp fragment yield as determined on a Bioanalyzer/Tapestation trace (i.e. % Integrated in trace is less than 5%).

To minimize adapter dimers, keep adapter ligation reagents on ice during ligation setup. If adapter dimers are >5% of your library, they should be removed from libraries as outlined here.

Figure. Example TapeStation trace from CUT&RUN H3K27me3 library containing an adapter dimer peak (~125 bp, red arrow) and expected library peak (~300 bp, blue arrow). Red lines denote the 200-700 bp range, used to determine library concentration.

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