Abstract 4731: Novel epigenetics technology for high-throughput processing of limited samples to study cancer using cavitation-based pixelated ultrasound and tagmentation-indexing ChIP-Seq
Authors: Sen Rwik , Tran Ngoc , Traynor Sarah , Maina Eric , Poole Jason 
Published: April 2023

Epigenetics has shown great potential in translational, clinical, and precision medicine research, but with inadequate implementation. This is due to major problems with sample availability, and processing which is laborious, costly, slow, inconsistent, and leads to sample loss. Epigenetics studies need samples with certain minimum concentrations and volumes to address all necessary questions. The samples often need to be in single cell or nuclear suspensions. For chromatin immunoprecipitation (ChIP), a consistent size of approximately 300 base pairs of DNA is essential but difficult to achieve. To address the above problems, we present novel cutting-edge epigenetics methodologies. Homogenization of various tissue types from diverse patients with different disease states is the first crucial step in sample processing. Hence, we present new protocols which are compatible with high-throughput cavitation-based pixelated ultrasound sonication. It processes limited quantities of samples in a 96-well plate where cells can be grown and antibody can be coated for ChIP. Further, 1 to 12 columns of the 96-well plate can be flexibly regulated to extract lysates. Our results show how this rapid and simple technology integrates with ChIP-sequencing (seq) and RNA-seq workflows, yielding very consistent output from limited tissue samples. Bioinformatics analysis of the ChIP-seq shows the corresponding biological significance. Certain limitations of ChIP has led to the evolution of other methods but they are mostly unsuccessful in mapping transcription factors. Hence, we next present a modified ChIP-seq combined with tagmentation, indexing and pooling, coupled to pixelated ultrasound based-cavitation. Here, individual samples are placed in each well of a 96-well plate containing unique indexing adapters fused to Tn5 transposase. Hence, each sample undergoes tagmentation - a process where DNA gets fragmented and simultaneously inserted with index adapters. Next, pixelated ultrasound helps to rapidly extract the complex of protein-tagmented DNA from 96 samples which are pooled into a single tube because they are uniquely indexed, then split up for immunoprecipitation by different antibodies. Bioinformatics analysis shows that the results are robust, consistent, and match ENCODE datasets. Hence, our methods can help to process high-throughput samples for AACR Project GENIE. Overall, we present novel technology to process limited human samples for enabling epigenetics research at the clinical interface from bench to bedside. The simple and less expensive methods yield high quality samples to enable high-throughput analysis of epigenetics including transcription factors which are important in cancer. They help us to understand targeted epigenetic regulations of genes for therapeutic developments and treating cancer.

Citation Format: Rwik Sen, Ngoc Tran, Sarah Traynor, Eric Maina, Jason Poole. Novel epigenetics technology for high-throughput processing of limited samples to study cancer using cavitation-based pixelated ultrasound and tagmentation-indexing ChIP-Seq. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4731.