High-spatial-resolution ECM-inclusive multi-omics sequencing of human PFA and FFPE tissue slides

摘要 该项目的重点是加速开发高空间分辨率的测序技术，用于 通过组织中的确定性条形码进行转录和蛋白质组的共同映射(hsrtp-seq)，这将是 用多聚甲醛(PFA)固定和福尔马林固定石蜡包埋(FFPE)的人体组织验证 标本。与任何现有的空间组学技术相比，这是一种全新的方法。这个 核心思想是使用一种新的技术对组织中的RNA、蛋白质或其他生物分子信息进行分子条形码 微流控原位条码方法。条形码后的组织载玻片在形态上保持完整，但 由组织像素的马赛克组成，每个像素都有一个不同的DNA条形码。像素的大小为 小到~5-10μm，接近单个细胞的大小。它是建立在Illumina Next的力量之上的 代测序(NGS)系统，实现显著更高的样本高通量、更低的成本和 消除重复单分子成像的繁琐程序，如在seqFISH中。它将展示 高空间分辨率(约5-10μm像素大小)、高吞吐量(每天多达100个组织样本流动条形码 每个操作者)和高含量(全基因组mRNA、蛋白质和非细胞环境)。使用面板 在针对细胞外基质(ECM)蛋白的DNA标记抗体中，这种方法进一步允许空间 组学测序包括非细胞成分的图谱，这些成分在scRNA中完全缺失 SEQ或当前的空间转录组技术。它是唯一适合于绘制人类胶原蛋白的地图 组织，包括心脏，主动脉，皮肤和肾脏，以提高我们对细胞外基质在正常 生理、疾病和衰老。我们将追求以下具体目标。在UG3阶段，我们将开发一个 一套新的设备显著增加了组织图谱的面积(4 mm×4 mm)，发展了蛋白质组规模 (~500个蛋白质共同分析)和包含ECM的空间测序，并开发了一种新的组织优化 在相同的组织切片上进行hsrtp-seq，并生成一组3D空间转录组- 来自人类心脏或主动脉的蛋白质组图谱数据。在UH3阶段，我们将进一步开发一种多针注射 Head以增加样本吞吐量(每天最多100个样本)和测绘区域(1.2cmx1.2 cm) 进一步扩大和自动化，开发一种新的组织内模板切换方法以保留完整的组织 Hsrtp-seq之后的部分，用于在同一组织载玻片上进行其他测量并构建3D 组织图谱，并最终开发了一种优化的PFA和FFPE组织方案来生成3D多组学 来自人类心脏、主动脉、皮肤和肾脏的组织图谱数据(每个样本20个组织切片)。

SUMMARY This project focuses on the accelerated development of a high-spatial-resolution sequencing technology for the co-mapping of transcriptomes and proteomes (hsrTP-seq) via deterministic barcoding in tissue, which will be validated with paraformaldehyde(PFA)-fixed and formalin-fixed paraffin-embedded (FFPE) human tissue specimens. This is a fundamentally new approach as compared to any existing spatial omics technologies. The core idea is to molecularly barcode RNAs, proteins, or other biomolecular information in tissues using a novel microfluidic in situ barcoding method. The tissue slide after barcoding remains morphologically intact but consists of a mosaic of tissue pixels, each of which has a distinct DNA barcode. The size of the pixels is as small as ~5-10μm, which is close to the size of individual cells. It is built upon the power of Illumina’s Next Generation Sequencing (NGS) systems to achieve significantly higher sample high-throughput, lower cost, and the elimination of laborious procedures for repeated single-molecule imaging as in seqFISH. It will demonstrate high-spatial-resolution (~5-10μm pixel size), high-throughput (up to 100 tissue samples flow barcoded per day per operator), and high-content (genome-wide mRNAs, proteins, and non-cellular environment). Using a panel of DNA-tagged antibodies against extracellular matrix (ECM) proteins, this approach further allows for spatial omics sequencing to include the mapping of non-cellular components, which are completely missing in scRNA- seq or current spatial transcriptomics technologies. It is uniquely suited for mapping human collagenous tissues including heart, aorta, skin, and kidney to improve our understanding of the role of ECM in normal physiology, disease and aging. We will pursue the following specific aims. In the UG3 phase, we will develop a set of new devices to significantly increase the tissue mapping area (4mmx4mm), develop a proteome-scale (~500 proteins co-analyzed) and ECM-inclusive spatial sequencing, and develop a novel tissue optimization protocol performed on the same tissue slide for hsrTP-seq, and generate a set of 3D spatial transcriptome- proteome atlas data from human heart or aorta. In the UH3 phase, we will further develop a multi-pin injection head to increase sample throughput (up to 100 samples per day) and the mapping area (1.2cmx1.2cm) for further scale up and automation, develop a new in-tissue template switching method to retain intact tissue section after hsrTP-seq for conducting other measurements on the same tissue slide and constructing 3D tissue atlas, and finally develop an optimized PFA and FFPE tissue protocol to generate the 3D multi-omics tissue atlas data (>20 tissue sections per sample) from the human heart, aorta, skin, and kidney.