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. Overall design: Naïve splenic follicular B cells were isolated from mice and stimulated in culture for 48 hours with 2 µg/mL anti-CD40 and 10 ng/mL IL-4. Cells were collected and processed for CUT&RUN at varying amounts of nuclei and on freshly isolated culture cells or cultured cells that were frozen and thawed. Target antibodies used were H3K4me3, IgG, RNA Pol II