Antibodies, Automation, and the Fight for Reproducibility (Alon Goren)

In this episode we speak with Alon Goran from UC San Diego about his work at the intersection of genomic technology development and chromatin biology. We discuss how his lab studies how the epigenome is regulated, how disruption of that regulation contributes to disease, and how technology can be improved to make results more robust and reproducible.

We talk about his early interest in biology, how that developed through medical research training, and how a molecular biology lab shaped the direction of his career. He explains how curiosity about how cells and organisms work led him toward genomics and chromatin research.

We then discuss several methods from his career, including early direct sequencing approaches for small amounts of DNA and RNA, ChIP-based methods for chromatin regulators, and work on improving ChIP-seq workflows. He explains why antibody choice matters, why monoclonal antibodies can improve reproducibility, and how automation helped scale the process.

We also cover his work on spike-in normalization, including the risks of using exogenous chromatin incorrectly and the need for better safeguards in genome-wide comparisons. He describes a newer approach that uses two spike-ins to provide multiple checks on normalization.

Finally, we discuss his work on short tandem repeats, zebrafish heart regeneration, and SIRT6-related polymerase pausing, as well as a newer platform that converts molecular interactions into sequencing-readable barcodes. He closes by stressing the importance of validation, careful protocol design, and methods that can be used reliably by multiple people.

References

• Ram, O., Goren, A., Amit, I., Shoresh, N., Yosef, N., Ernst, J., Kellis, M., Gymrek, M., Issner, R., Coyne, M., Durham, T., Zhang, X., Donaghey, J., Epstein, C. B., Regev, A., & Bernstein, B. E. (2011). Combinatorial patterning of chromatin regulators uncovered by genome-wide location analysis in human cells. Cell, 147(7), 1628–1639. https://doi.org/10.1016/j.cell.2011.09.057
• Busby, M., Xue, C., Li, C., Farjoun, Y., Gienger, E., Yofe, I., Gladden, A., Epstein, C. B., Cornett, E. M., Rothbart, S. B., Nusbaum, C., & Goren, A. (2016). Systematic comparison of monoclonal versus polyclonal antibodies for mapping histone modifications by ChIP-seq. Epigenetics & chromatin, 9, 49. https://doi.org/10.1186/s13072-016-0100-6
• Patel, L. A., Cao, Y., Mendenhall, E. M., Benner, C., & Goren, A. (2024). The Wild West of spike-in normalization. Nature biotechnology, 42(9), 1343–1349. https://doi.org/10.1038/s41587-024-02377-y
• Ben-Yair, R., Butty, V. L., Busby, M., Qiu, Y., Levine, S. S., Goren, A., Boyer, L. A., Burns, C. G., & Burns, C. E. (2019). H3K27me3-mediated silencing of structural genes is required for zebrafish heart regeneration. Development (Cambridge, England), 146(19), dev178632. https://doi.org/10.1242/dev.178632

Related Episodes

• Taking ChIP from Yeast to ENCODE to Enable Genome-Wide Regulatory Protein Mapping (Peggy Farnham)
• Comparing CUT&Tag to ENCODE ChIP-Seq in Alzheimer's Disease Samples (Sarah Marzi)
• Chromatin Profiling: From ChIP to CUT&RUN, CUT&Tag and CUTAC (Steven Henikoff)

Contact

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• Email: podcast@activemotif.com