Chromatin profiling from formalin-fixed paraffin-embedded samples for biomarker discovery

As the oncology field moves towards a precision medicine model of patient care, understanding and profiling the epigenetic landscape is increasingly important. Gene expression and cell function are regulated by epigenomic features, including histone post-translational modifications (PTMs), transcription factors, and other chromatin proteins. Mapping the location of these features provides a powerful approach to study chromatin mechanisms driving disease and can be leveraged for biomarker and drug development. Formalin-fixed paraffin-embedded (FFPE) tissues banked from cancer clinical trials could be a rich resource for retrospective biomarker studies, due to their association with drug response and disease progression data. However, using FFPE tissues for genomic mapping assays has been technically challenging for multiple reasons. For instance, the removal of paraffin, heavy cross-linking conditions, and degraded nucleic acids can make FFPE samples unsuitable for standard transcriptomic and epigenomic mapping assays (RNA-seq, ATAC-seq, ChIP-seq). To better leverage chromatin profiling for translational research, we developed a modified CUT&Tag workflow that is compatible with FFPE samples (CUT&Tag-FFPE). CUT&Tag (Cleavage Under Targets and Tagmentation) is an immunotethering-based strategy that uses pAG-Tn5 to selectively cleave and ligate sequencing adapters at antibody-bound chromatin. The entire assay takes place on-slide with intact cells or nuclei extracted from scrolls, enabling a highly streamlined workflow. In this study, we optimized every major step of the workflow, including paraffin removal, in situ tissue permeability or nuclei extraction, crosslink removal, antibody selection, and tagmentation. We have applied these modifications to successfully map transcription-linked open chromatin across a variety of FFPE sample tissue types from mouse and human. We find that CUT&Tag-FFPE correlates with RNA-seq and ATAC-seq from corresponding fresh, frozen material. Additionally, we observed open chromatin at tissue specific promoters and at distal elements, such as enhancers. By enabling insights into genomic regulatory mechanisms from FFPE biorepositories, CUT&Tag-FFPE has the potential to accelerate biomarker discovery and drug development research for precision oncology.