Do you have any tips for the preparation of good CUT&RUN DNA sequencing libraries?

Yes, there are a lot of tips for CUT&RUN DNA library preparation, especially for CUT&RUN assays with an extremely low number of cells. Compared to ChIP-DNA samples, CUT&RUN DNA has two unique features. First, it is smaller in size and, secondly, the yield of DNA is much lower due to fewer starting cells being used in a CUT&RUN assay.

Because CUT&RUN DNA is small, you need to decrease the incubation temperature from 65C to 50C during end prep and A tailing to prevent the generation of ssDNA. Also, because of its smaller size, you may benefit from using 1.1X volumes instead of 0.9X volumes of Ampure beads during library DNA purification. Importantly, the extension time during PCR amplification of the library can be decreased from 75s to 10-15s. This not only saves time during the PCR program, but it also excludes the possibility of large non-specific DNA being amplified.

Since CUT&RUN DNA has a low yield, you will also need to add less adaptor DNA for ligation in order to avoid adaptor contamination in the final library sample. You may also need to increase the PCR cycles up to 20 in order to get enough library DNA, and at the same time you can decrease the final volume of TE buffer to elute your library DNA from the beads.

We have listed all of our recommendations for CUT&RUN DNA library prep in our CUT&RUN assay kit protocol, for your reference.
 

Last updated: September 12, 2024