The main difference between CUT&RUN and ChIP-seq is how they capture target-enriched chromatin, which influences cell number requirements, sequencing depths, data quality, and assay throughput. Importantly, both assays use antibodies specific to a given target, which can include histone PTMs, transcription factors, and other chromatin-associated proteins. In ChIP-seq, or chromatin immunoprecipitation sequencing, an antibody is used enrich or “pull-down” targets from a large pool of fragmented chromatin. Generating the chromatin pool requires cross-linking, cell lysis, and chromatin fragmentation, each of which must be carefully optimized and controlled to reduce artifacts and background signal. Following fragmentation, bead-coupled antibodies are used to pull down target fragments. Cross-links are reversed and DNA is purified for sequencing analysis. Chromatin immunoprecipitation (the IP step) is inherently noisy. In addition to problems with antibody performance, the magnetic beads may pull down off-target fragments along with the chromatin containing the target. IP also requires highly stringent washes, which can reduce yields and lower signal-to-noise.
CUT&RUN revolutionized the study of chromatin regulation by enabling targeted release of genomic fragments into solution. The streamlined workflow does not require sonication or IP steps, resulting in dramatically reduce background and improved on-target recovery. The result is an assay amenable to greater experimental throughput, allowing deeper and more rapid investigations to uncover epigenetic biology.
Specifically, compared to ChIP-seq, CUT&RUN features:
Lower cell requirements. CUTANA™ CUT&RUN assays generate high-resolution profiles using as few as 5,000 cells, and lower cell numbers have been reported in the literature (PubMed PMIDs: 30955888, 33911257).
Improved signal-to-noise. Separation of target-bound chromatin from bulk material means lower background and robust signal – even when using fewer cells.
Reduced cost. High signal-to-noise enables confident peak calling with ~10-fold fewer sequencing reads and less primary antibody.
Less optimization. The entire assay is performed in intact cells (or nuclei) immobilized on a solid support. These advances eliminate the most technically challenging steps of ChIP-seq, including cell lysis, chromatin fragmentation, and IP.
Faster. CUT&RUN provides a user-friendly, streamlined protocol (or kit) that allows scientists to go from cells to sequencing data in less than four days (vs. weeks for ChIP-seq). CUT&RUN can also be automated for greater throughput.