Session: 803. Emerging Tools, Techniques and Artificial Intelligence in Hematology: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Translational Research, emerging technologies, Technology and Procedures, omics technologies
In our study, we implemented Covaris’s Adaptive Focused Acoustics® (AFA®) technology to miniaturize sample volumes for both chromatin shearing and immunoprecipitation in a 96-well format. This approach enables a standardized, robust, automatable, half-day workflow for both ChIP and Hi-ChIP applications. AFA ChIPseq- was applied to flow sorted hematopoietic stem and progenitor cells (HSPC) from individual mice. We utilized a range of epitopes including major histone modifications (H3K9me1/2/3, H3K27ac) and factors (CTCF, cohesin). Interestingly, we found epitope-dependent differences in AFA-enhanced binding with CTCF saturating at 30 minutes, whereas H3K27ac required 4 hours of AFA pulse to facilitate chromatin binding. Additionally, we evaluated low input in vitro and in vivo samples, with limited dilution of lineage negative HSPCs ranging from 500,000 to 5,000 cells with comparable sheared chromatin yield and immunoprecipitated material. Overlap of peaks called by MACS2 was consistent among the various cell inputs as well as when compared with gold standard CTCF ChIPseq- data previously performed on pooled samples from our previously published work. The low cell input capability permitted deconvolution of dynamic epigenetic changes in protein binding and chromatin structure during normal lineage specification of hematopoietic stem cells. Epigenetic remodeling of these sites was consistent with established gene programs essential for myeloid differentiation. Data were found to be in alignment with standard ChIPseq- and Hi-C datasets generated from similar cell populations.
The simplified and significantly shortened (Hi)ChIP-seq processes showcased in this study will be highly beneficial for both research and clinical diagnostic applications owing to their requirements for using 96-well plate format and their ability to become integrated into automated workflows. Taken together, this technology and workflow represents a high throughput epigenetic system for small cell numbers of cells with a multitude of applications enabling laboratories focused in cell biology and translational research.
Disclosures: O'Hare: Covaris: Current Employment. Garding: Covaris: Current Employment. Viny: Nooma Bio: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Arima Genomics: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.
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