A low-input compatible, end-to-end kitted HiChIP workflow for concurrent analyses of transcriptional protein binding and chromatin interactions toward a mechanistic understanding of gene regulation

低输入兼容的端到端配套HiChIP工作流程，用于转录的并发分析 蛋白质结合和染色质相互作用对基因调控机制的理解 Arima Genomics 项目总结/摘要 基因表达的精确调节对于建立细胞特性至关重要，而基因的错误调节 导致人类疾病。细胞利用调控元件（RE）、短DNA序列来调节基因表达 嵌入整个基因组，谁是转录蛋白结合，以促进其调节 功能分子图谱工具，如染色质免疫沉淀和下一代测序（ChIP- seq），沿着基因组产生RE的“图谱”，并且已经成为理解基因的普遍方法。 基于唯一的RE签名来调节和定义小区类型和状态。然而，RE的这些位置是 只有在线性基因组的背景下才能理解。实际上，RE在三分钟内执行它们的基因控制， 三维基因组。因此，要真正了解基因调控-基因调控必须映射在 3D.事实上，开发了高通量染色质相互作用捕获（HiC）以产生3D相互作用图 人类基因组中30亿个碱基的总和。HiC促进了DNA的几个基本原理的发现 3D折叠，包括DNA错误折叠导致疾病的情况。然而，HiC确实测量了 转录蛋白结合，也不知道染色质相互作用是否是调节性的，因此在转录蛋白结合中具有有限的效用。 推进我们对3D基因调控的理解。最近，新的方法尝试将分子联合收割机 ChIP-seq和染色质相互作用捕获的步骤，以测量转录蛋白结合和介导的 单次测定中的染色质相互作用。然而，这些方法，称为ChIA-PET和HiChIP，不 分别有效地捕获染色质相互作用或转录蛋白结合。因此， 需要改进方法以真正促进3D基因调控作图。 我们通过高度优化的第一代HiChIP解决方案Arima-HiChIP（A-HiChIP）来满足这一未满足的需求， 这证明了转录蛋白结合和染色质的有效和可重复的映射， 在细胞系中的相互作用，具有更高的细胞输入和有限的转录蛋白。我们的团队已被 在染色质相互作用捕获及其商业化科学方面无与伦比的专业知识。一是 在2018年商业化的Arima-HiC试剂盒，用于研究染色质相互作用的一般原理，并生成 商业化第一年收入120万美元，第二年收入200万美元，拥有500多个客户， 从2018年到2019年增长100%。基于VOC分析，我们将重点转移到开发更相关的 我们将产品推向基因调控市场- A-HiChIP -这是客户想要的，代表了一个更大的市场 机会事实上，在我们自筹资金的第一阶段研发和商业开发之后，我们推出了第一个 第二代HiChIP解决方案进入市场，并取得了显着的成功-通过HiChIP不断增长来衡量 从19%到40%的收入贡献，提高收入质量，吸引KOL，大 财团和新型冠状病毒研究然而，这些工具包在功能方面是有限的-它们并不强大 对于一系列转录蛋白，它们并没有针对组织样品进行优化， 降低样品输入量。为了实现更广泛的采用和发现，我们展示了 我们的第二代A-HiChIP解决方案的发展，向低样品输入的进步， 组织和更广泛的转录蛋白质。我们在提供的内部样品上验证了该技术 通过学术合作者和外部客户手中通过原型测试套件。 作为这个直接2阶段II计划的一部分，我们建议进一步发展我们的技术， 兼容的端到端配套HiChIP解决方案，用于同时分析转录蛋白结合和 染色质的相互作用在组织样本和跨主机的重要转录蛋白质。我们亦建议 严格和必要的产品开发实验，以确保一个强大的，优质的商业化- 基于性能套件的产品，以最佳方式集成到生态系统中。成功完成后 目标1和2的技术和商业发展，我们建议对我们的下一个- 通过协作和原型（beta）套件以及生物信息学评估， 意见领袖（KOL）在客户群。

A low-input compatible, end-to-end kitted HiChIP workflow for concurrent analyses of transcriptional protein binding and chromatin interactions toward a mechanistic understanding of gene regulation Arima Genomics Project Summary/Abstract Precise regulation of gene expression is paramount to establishing cellular identities, and mis-regulation of genes causes human disease. Cells regulate gene expression using regulatory elements (REs), short DNA sequences embedded throughout the genome, who are bound by transcriptional proteins to facilitate their regulatory function. Molecular mapping tools, such as Chromatin immunoprecipitation and next gen sequencing (ChIP- seq), produce “maps” of REs along the genome and have been a ubiquitous approach towards understand gene regulation and define cell types and states based on unique RE signatures. However, these locations of REs are only understood in context of a linear genome. In reality, REs execute their gene control within a three dimensional (3D) genome. Therefore to truly understand gene regulation – gene regulation must be mapped in 3D. Indeed, high throughput chromatin interaction capture (HiC) was developed to produce 3D interaction maps of all 3 billion bases in the human genome. HiC has facilitated discovery of several fundamental principles DNA folding in 3D, including cases where DNA mis-folding contributes to disease. However, HiC does measure transcriptional protein binding, nor whether a chromatin interaction is regulatory, thus having limited utility in advancing our understanding 3D gene regulation. Recently, novel approaches attempt to combine the molecular steps of ChIP-seq and chromatin interaction capture to measure transcriptional protein binding and mediated chromatin interactions in a single assay. However these approaches, termed ChIA-PET and HiChIP, do not efficiently capture chromatin interactions or transcriptional protein binding, respectively. Therefore, there is dire need for improve methods that truly facilitate mapping of gene regulation in 3D. We satisfy this unmet need via our highly optimized, first generation HiChIP solution, Arima-HiChIP (A-HiChIP), that demonstrates efficient and reproducible mapping of transcriptional protein binding and chromatin interactions in cell lines, with higher cellular inputs and a limited set of transcriptional proteins. Our team has unmatchable expertise in the science of chromatin interaction capture and its commercialization. First, we commercialized Arima-HiC kits in 2018 for studying general principles of chromatin interactions and generated $1.2M in revenue in the 1st year of commercialization and $2M in revenue in the 2nd year, with 500+ customers, and 100% growth from 2018 to 2019. Based on VOC analytics, we shifted our focus to develop a more relatable product to the gene regulation market – A-HiChIP - that customers wanted and that represented a larger market opportunity. Indeed, after our self-funded phase-1 R&D and commercial developments, we launched our first generation HiChIP solution into the market and have seen remarkable success – measured by HiChIP growing from 19% to 40% of our revenue contributions, increased quality of revenue, and traction with KOLs, large consortia, and COVID research. However, these kits are limited in terms of the capabilities – they are not robust to a range of transcriptional proteins, they are not optimized towards tissue samples, and they are not optimized towards lower sample input quantities. To enable broader adoption and discovery, we have shown the development towards our second-generation A-HiChIP solution, with advancement towards low sample inputs, tissues, and a broader range of transcriptional proteins. We validate the technology on internal samples provided by academic collaborators and externally in customer hands via prototype beta kits. As part of this direct-2-phase II program, we propose to further develop our technology into truly robust, low input compatible, end-to-end kitted HiChIP solution for concurrent analysis of transcriptional protein binding and chromatin interactions in tissue samples and across a host of important transcriptional proteins. We also propose rigorous and essential product development experiments, to ensure commercialization of a robust, premium- performance kit-based product that is optimally integrated into the ecosystem. Upon successful completion of the technical and commercial developments in Aims 1 & 2, we propose to benchmark and validate the our next- generation HiChIP solution through collaboration and prototype (beta) kit and bioinformatics evaluations with key opinion leaders (KOLs) across customer segments.