In:
Cancer Research, American Association for Cancer Research (AACR), Vol. 82, No. 23_Supplement_2 ( 2022-12-01), p. A022-A022
Abstract:
Histone post-translational modifications (PTMs) play a critical role in chromatin regulation. It has long been suspected that the poorly understood capability of ‘PTM-specific’ antibodies (i.e., their specificity and efficiency) is a prime driver of the reproducibility crisis in biomedical research. Here we confirm the validity of this concern as it applies to epigenomic mapping studies. Extensive spike-in panels of PTM-defined DNA-barcoded nucleosome standards show that & gt;70% of & gt;500 commercial antibodies (and & gt;80% of the most highly cited) to & gt;50 histone lysine methyl and acyl states have failing performance in ChIP ( & gt;20% cross reactivity/ & lt;5% target recovery; www.ChromatinAntibodies.com). Variable lot behavior (of both polyclonals and monoclonals) shows the danger of focusing on catalog numbers without considering the inherent variability of biological reagents. Ultimately, these studies support the inclusion of in situ standards to control genomic mapping assays as an improved path out of this morass. Despite these advances, the application of ChIP-seq for cancer research has been hampered by its requirement for high cell inputs and low reliability. The recent development of immunotethering assays, such as Cleavage Under Targets and Release Using Nuclease (CUT & RUN), deliver high signal-to-noise mapping data using a fraction of the required cells compared to ChIP-seq. These innovations enable the application of epigenomics for cancer research, particularly for primary cells, sorted populations, and clinical samples where cell numbers are limited. By adapting our nucleosome spike-in control approach for CUT & RUN assays, we show that while the same antibody problems observed in ChIP-seq also pervade CUT & RUN, continuous use of spike-in controls improves assay rigor and reproducibility to realize the potential of CUT & RUN to advance cancer research. Citation Format: Andrea L. Johnstone, Danielle N. Maryanski, Keli L. Rodriguez, Ellen N. Weinzapfel, Bryan J. Venters, Katherine Novitzky, Matthew R. Marunde, Carolina P. Lin, Michael-Christopher Keogh. A field survey of histone PTM antibodies in epigenomic mapping approaches reveals widespread liabilities: Best practices and resources for reliable epigenetic studies. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A022.
Type of Medium:
Online Resource
ISSN:
1538-7445
DOI:
10.1158/1538-7445.CancEpi22-A022
Language:
English
Publisher:
American Association for Cancer Research (AACR)
Publication Date:
2022
detail.hit.zdb_id:
2036785-5
detail.hit.zdb_id:
1432-1
detail.hit.zdb_id:
410466-3
Permalink
