How do I pool CUT&Tag DNA libraries with a variety of yields?

Usually, the CUT&Tag DNA libraries from histone targets have a higher concentration than those from non-histone targets. We use the following formula to convert a library concentration from ng/µL to nM before diluting each library sample to the same concentration (nM) for pooling purposes: Concentration (nM) = 1,000,000 X Concentration (ng/µL) / library average size (bp) / 660. In addition to obtaining a greater library size for undetectable library samples from the Bioanalyzer or TapeStation system (as described in the above question), we would also suggest pooling the libraries that have a flat signal of 5-10 fold more than the libraries that show normal sized peaks on the Bioanalyzer or TapeStation systems. This ensures an even distribution of the number of reads among all samples. Usually, a library pool concentration of 2 nM DNA is enough for NGS purposes, although a higher concentration is always welcome.

Last updated: September 12, 2024