EpiCypher Inc.

Epigenetics refers to the study of chromatin structures and mechanisms that regulate gene expression independent of the underlying DNA sequence. Epigenetics and chromatin is a rapidly growing field of biomedical research, with implications in many diseases, including cancer, autoimmune diseases, and neurodegeneration. The base subunit of chromatin structure is the nucleosome, which consists of DNA wrapped around a histone octamer. Histone proteins are decorated with various post-translational modifications (or PTMs), which work together to form a “histone code” that regulates chromatin structure. Different combinations of PTMs have varying effects on gene expression, cell survival, and development of diverse human diseases, as noted above. Thus, accurate mapping of histone PTMs to the genome is key to understanding the role of chromatin structure in regulating gene expression and disease. There are several widely accepted methods researchers use to isolate and sequence chromatin marked with specific PTMs.

The most common method is chromatin immunoprecipitation, or ChIP, which uses antibodies to specific histone PTMs for enrichment of unique subsets of chromatin. The DNA associated with these histone PTMs then is isolated, sequenced, and mapped to the genome. However, one of the major problems that researchers experience when performing ChIP is that many histone PTM antibodies display poor specificity. Often, these antibodies bind multiple PTMs, in addition to their stated target, or they bind weakly to the target histone PTM (i.e. low enrichment). In addition, ChIP has high background, and requires enormous inputs (>500K cells) and sequencing depths (20-40 million reads per sample). Thus, although ChIP is a standard tool in the field, it is expensive, unreliable, and delivers low-quality data. New methods are desperately needed to improve the applications of chromatin to biomedical research and drug development.

How can EpiCypher CUTANA CUT&RUN assays improve your chromatin studies?
The recent development of chromatin immunotethering technologies has transformed chromatin profiling studies, providing access to high-resolution data with reduced cell input and sequencing depth requirements. EpiCypher has developed robust tools and protocols for the Cleavage Under Targets and Released Using Nuclease method (CUT&RUN) with the recent launch of CUTANA CUT&RUN assays.

In CUT&RUN, cells are immobilized onto a solid support, permeabilized, and treated with a factor-specific antibody (i.e. histone PTM). A protein A- protein G fusion is then used to “tether” a Micrococcal Nuclease to antibody-bound chromatin. This pAG-MNase is activated by the addition of calcium to cleave DNA at antibody-bound loci, and clipped fragments diffuse out of the cell into the supernatant. These fragments are collected and sequenced by standard next-generation sequencing methods.

By selectively cleaving and isolating the target chromatin of interest, the data generated using EpiCypher CUTANA assays have vastly improved signal-to-noise and reduced background compared to ChIP, while also using fewer sequencing reads (2-5 million) and cells (<500K).

EpiCypher offers recombinantly produced pAG-MNase from E. coli for use in your ChIC and CUT&RUN assays.

Visit EpiCypher.com for more information, including the latest CUTANA CUT&RUN protocol.