Internally Calibrated Chromatin Immunoprecipitation Using Barcoded Nucleosomes

DESCRIPTION provided by applicant Chromatin immunoprecipitation ChIP is one of the core methods in the study of epigenetic regulation of gene expression Analytical methods such as qPCR microarray and next generation sequencing NGS have increased throughput but not the reliability of ChIP datasets Using state of the art reagents ChIP is at best semi quantitative and at worst unrepresentative of in vivo post translational modification PTM density To address this short coming EpiCypher Inc has partnered with Dr Alex Ruthenburg of the University of Chicago who has pioneered a method to internally standardize ChIP experiments for subsequent normalization and quantitation of epigenetic marks The method referred to here as Internally Calibrated Immunoprecipitation Sequencing or ICeChIP Seq was recently licensed on an exclusive basis by EpiCypher Inc and relies on designer nucleosomes collaboratively developed and manufactured by EpiCypher and the Ruthenburg lab These nucleosomes are modified with an epigenetic mark of interest and wrapped in a DNA sequence containing a unique identifying barcode sequence Barcoded designer nucleosomes are added to the ChIP reaction at various concentrations containing purified chromatin and a bead attached pull down antibody against the epigenetic mark of interest After immunoprecipitation next generation sequencing data is analyzed for the number of reads detected for each barcode and immunoprecipitated input DNA with the resulting ratio used to compute IP enrichment quantitatively Read number can then be normalized to input concentration for each barcoded nucleosome providing a standard curve for quantitation of sample DNA reads The barcoded nucleosomes serve as calibrators because they are subjected to the same sources of variability the sample chromatin experiences during the ChIP reaction and they precisely resemble the target of the IP Proof of concept development of the assay has focused on the following synthesis of barcoded nucleosomes analysis of their quality development of an H K me assay analysis algorithms characterizing signal to noise ratio minimum chromatin input demands and day to day variability Given the strong data generated to date and the urgent need for an improved ChIP Seq assay we have proposed a Direct to Phase II workplan toward the commercialization of this powerful method The Phase I report provides compelling evidence that the method is a dramatic improvement over existing methods with Phase II aims focused on manufacturing a kit for the H K me assay reagents external validation of this assay and expanding the menu of PTM specific ICeChIP Seq reagents to other epigenetic marks PUBLIC HEALTH RELEVANCE Chromatin immunoprecipitation ChIP is a cornerstone method to investigating epigenetic regulation of gene expression a currently expanding area of research toward new therapeutics for cancer and other indications ChIP however is a non quantitative method providing investigators only with information regarding the presence or absence of various protein chromatin interactions with no data regarding abundance even on a relative basis Currently reproducibility issues prohibit clinical applications The method under development here employs nucleosome based controls which can be added to ChIP reactions and uniquely detected using next generation sequencing methods to provide a standard curve thereby allowing a quantitative method referred to here as Internally Calibrated Immunoprecipitation Sequencing or ICeChIP Seq