High-throughput single-cell co-sequencing of small and large RNAs to identify molecular circuitry in cancer

项目总结 小RNA(SmRNAs)是一种重要的非编码调节因子，广泛参与人类癌症的发生和发展 进步。在肿瘤研究中，对smRNAs的功能和机制进行了广泛的研究。他们是 也被作为新的治疗靶点进行研究，但到目前为止结果并不令人满意，部分原因是 人类癌症中miRNA-mRNA调控网络的复杂性与细胞的异质性。因此， 可以在大量单个细胞中共同分析smRNA和大RNA(LgRNAs)的新工具可能会有所帮助 解决这一长期存在的挑战，为smRNA研究增加一个新的维度，增强对 小分子RNA参与癌症生物学的新机制，并使其在癌症中的潜在应用成为可能 诊断学和治疗学。Pis Fan和Lu之间的密切合作首次展示了 时间，来自相同单个细胞的smRNA和lgRNA的共同测序(Wang等人，自然通讯， 2019年)。它进一步表明，在单细胞水平上拥有配对的smRNA和lgRNA图谱可以揭示 MiRNA介导的基因调控及其调控miRNA表达和细胞间的新机制 异质性。然而，这种单细胞smRNA-lgRNA共测序技术是一个手动过程， 低吞吐量和高成本，由于统计能力不足而限制了其在癌症研究中的潜力 询问高度异质性的肿瘤细胞。在这一应用中，我们建议对此进行高级开发 提供高通量单细胞smRNA/lgRNA共测序(scSLRco-seq)技术。它 采用新的滑移微器件与新的分子条形码方案相结合，并 下游生化同时分析1000‘S单细胞中的sMRNA和lgRNAs。 具体地说，我们将(目标1)开发一种微流控交叉流动图案化方法来创建2500个DNA条形码 用于小RNA和大RNA的空间耦合捕获和条形码的阵列(AIM2)开发了一种新的滑动- 用于集成多步骤生化工作流程以实现高通量scSLRCo-seq的转移芯片，以及(Aim3) 使用人类和小鼠髓系白血病模型验证这项技术。这项新技术解决了 缺乏高通量单细胞smRNA/lgRNA联合图谱的能力，填补了单细胞组学的空白 并使研究以前无法回答的新问题成为可能。它代表着世界上 并将在癌症研究和应用中得到广泛应用。

PROJECT SUMMARY Small RNAs (smRNAs) are important non-coding regulators broadly implicated in human cancer initiation and progression. The functions and mechanism of smRNAs have been widely studied in cancer research. They are also being investigated as new therapeutic targets but the results so far are unsatisfactory, due in part to the complexity of miRNA-mRNA regulatory network coupled with cellular heterogeneity in human cancers. Thus, new tools that can co-profile smRNAs and large RNAs (lgRNAs) in large numbers of single cells can help address this long-standing challenge, add a new dimension to smRNA research, empower the discovery of new mechanisms of smRNAs to participate in cancer biology, and enable potential applications to cancer diagnostics and therapeutics. A close collaboration between PIs Fan and Lu has demonstrated, for the first time, co-sequencing of both smRNAs and lgRNAs from the same single cells (Wang et al., Nature Comm., 2019). It further showed that having paired smRNA and lgRNA profiles on the single-cell level can reveal miRNA-mediated gene regulation and new mechanisms for controlling miRNA expression and intercellular heterogeneity. However, this single-cell smRNA-lgRNA co-sequencing technology is a manual process with low throughput and high cost, limiting its potential for cancer research due to insufficient statistic power to interrogate highly heterogeneous tumor cells. In this application, we propose the advanced development of this technology to deliver a high-throughput single-cell smRNA/lgRNA co-sequencing (scSLRco-seq) technology. It employs a novel slip-transfer microdevice in combination with novel molecular barcoding scheme and downstream biochemistry for simultaneous analysis of smRNAs and lgRNAs in 1000’s of single cells. Specifically, we will (Aim 1) develop a microfluidic cross-flow patterning method to create 2,500 DNA barcode arrays for spatially-coupled capture and barcoding of small and large RNAs, (Aim2) develop a novel slip- transfer chip to integrate multi-step biochemistry workflow for high throughput scSLRCo-seq, and (Aim3) validate this technology using human and mouse myeloid leukemia models. This novel technology addresses the lack of capability for high-throughput single-cell smRNA/lgRNA co-profiling, filling a gap in single-cell omics field and enabling the study of new questions previously impossible to answer. It represents a major leap in the field and will find wide-spread use in cancer research and applications.