Optimized CUT&RUN protocol for a non-histone protein in activated primary mouse B cells

ChIP-seq has long been the standard for study of chromatin-protein interactions. However, development of a new technique, CUT&RUN, shows substantial advantages compared to ChIP-seq including higher quality signal while using substantially less sample. While a powerful technique, the original protocol was unsuitable for obtaining high-quality data for non-histone proteins from activated primary B lymphocytes. To adapt this protocol for B cells, cells were fixed prior to nuclear isolation, and several adjustments were introduced to the procedure and reagents. We measured binding of RNA polymerase II to five genes and used binding of H3K4me3 histone as a positive control. Robust peaks in transcribed genes were detected with as little as 100k nuclei. Additionally, freeze-thaw of B cells prior to processing did not influence results, emphasizing the flexibility of this modified technique. Using the protocol described here will allow one to quantify low abundance proteins bound to DNA from limited numbers of B cells with more efficiency than can be achieved from the current standard, ChIP-seq.