# Development of novel spike-in controls for quantitative chromatin-associated protein profiling

> **NIH NIH R44** · EPICYPHER, INC. · 2020 · $978,257

## Abstract

PROJECT SUMMARY
 Alterations in chromatin regulators are associated with diverse human pathologies. The ability to
quantitatively assess these factors in healthy and diseased cells is essential to accelerate the development of
therapeutics targeting epigenetic regulation (a growing area of study, with many candidates already in clinical
trials). However, ChIP-Seq, the most widely-used approach to map the genomic location of Chromatin
Associated Proteins (ChAPs), is often limited by poor resolution, sensitivity, and reliability. Dr. Steven Henikoff’s
group recently developed CUT&RUN (Cleavage Under Targets and Released Using Nuclease), a new mapping
approach with vastly improved assay performance vs. ChIP-Seq. CUT&RUN uses ChAP-targeting antibodies to
locally tether protein A-micrococcal nuclease (pA-MNase) to chromatin in intact nuclei, followed by controlled
MNase activation to cleave nearby DNA. Sequencing of the subsequently released DNA fragments yields precise
target localization profiles using fractions (vs. ChIP-Seq) of the required cellular input (100-fold less) and
sequencing depth (>10-fold less). The efficiency of this method could now enable pre-clinical applications in a
high-throughput format, such as quantifying the genome-wide effects of epigenetic therapeutics. However,
delivering on such promise will require the development of quantitative spike-ins.
 In this Fast-Track SBIR proposal, EpiCypher® is partnering with Dr. Kami Ahmad of the Henikoff
lab to develop quantitative spike-in controls for ChAPs using CUT&RUN (ChAP-CUT&RUN). EpiCypher
has recently developed the application of DNA-barcoded recombinant designer nucleosomes (dNucs) as
quantitative spike-in controls for histone post-translational modification (PTM) ChIP studies (i.e. SNAP-ChIP®).
However, there are no tools to normalize mapping data for ChAPs, which make up the largest segment of the
ChIP-Seq market. The innovation of this project is the engineering of DNA-barcoded dNucs that contain
either: 1) a ChAP epitope; or 2) a Short Peptide Tag (SPT; e.g. FLAG) fused to the N-terminus of histone
H3. These can then be used to capture ChAP- or SPT-specific antibodies (both commonly used for ChAP
mapping studies) in a CUT&RUN workflow. In Aim 1 (Phase I), we will develop a set of DNA-barcoded dNuc
spike-ins for quantitative analysis of ChAPs (e.g. CTCF [transcription factor] and BRD4 [chromatin interactor]).
Phase I will be successfully completed when we use these dNucs in CUT&RUN for quantitative sample
normalization. In Aim 2 (Phase II), we will expand / scale manufacturing of ChAP-CUT&RUN spike-in control
panels and apply these reagents to establish robust workflows for quantitative sample normalization genome-
wide. In Aim 3 we will develop and externally validate ChAP-CUT&RUN beta kits. We envision ChAP-CUT&RUN
will become one of the most widely used assays in the epigenetics field (given the vast gain in assay metrics vs.
ChIP-Seq), with the potential to open new mark...

## Key facts

- **NIH application ID:** 10219746
- **Project number:** 4R44DE029633-02
- **Recipient organization:** EPICYPHER, INC.
- **Principal Investigator:** Michael-Christopher Keogh
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $978,257
- **Award type:** 4N
- **Project period:** 2019-09-13 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10219746

## Citation

> US National Institutes of Health, RePORTER application 10219746, Development of novel spike-in controls for quantitative chromatin-associated protein profiling (4R44DE029633-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10219746. Licensed CC0.

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