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

对人类 PFA 和 FFPE 组织切片进行高空间分辨率 ECM 多组学测序

• 批准号: 10687349
• 负责人: Rong Fan
• 金额: $ 60万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-10 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10687349
• 关键词: 3-Dimensional; Adopted; Adoption; Aging; Antibodies; Aorta; Area; Atlases; Automation; Bar Codes; Biomedical Engineering; Cardiac; Cardiovascular system; Cells; Communities; Computer Analysis; DNA; Data; Data Set; Databases; Development; Devices; Discrimination; Disease; Embryo; Environment; Extracellular Matrix; Extracellular Matrix Proteins; Fluorescent in Situ Hybridization; Formalin; Freezing; Generations; Genes; Genomic approach; Glass; Head; Health; Heart; Histology; Human; Human BioMolecular Atlas Program; Human body; Image; In Situ; Individual; Injections; Kidney; Longevity; Manuals; Measurement; Messenger RNA; Methods; Microfluidics; Molecular; Morphology; Motivation; Mus; Nature; Organ; Organogenesis; Paraffin Embedding; Phase; Physiology; Preparation; Procedures; Process; Proteins; Proteome; Protocols documentation; Public Health; RNA; Research; Research Personnel; Resolution; Role; Sampling; Skin; Slide; Solid; Specimen; Spottings; Surgeon; System; Technology; Tissue Embedding; Tissue Sample; Tissue atlas; Tissue imaging; Tissues; Validation; Variant; base; cost; extracellular; genome-wide; high throughput technology; human data; human tissue; improved; interest; molecular imaging; monolayer; multiple omics; next generation sequencing; novel; novel strategies; paraform; process optimization; rheumatologist; scale up; single molecule; single-cell RNA sequencing; synergism; tissue mapping; tissue mosaicism; transcriptome; transcriptome sequencing; transcriptomics

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.

概括 该项目着重于加速的高空间分辨率测序技术的发展 转录组和蛋白质组织（HSRTP-SEQ）通过组织中的确定性条形码进行绘制，这将是 用多聚甲醛（PFA）固定和福尔马林固定的石蜡（FFPE）人体组织验证 标本。与任何现有的空间OMIC技术相比，这是一种从根本上开始的方法。 核心思想是使用新颖的组织中的分子条形码RNA，蛋白质或其他生物分子信息 微流体原位条形码方法。条形码后的组织滑动在形态上保持完整，但 由组织像素的镶嵌物组成，每个像素都有不同的DNA条形码。像素的大小为 小为〜5-10μm，接近单个细胞的大小。它建立在Illumina的下一个力量之上 生成测序（NGS）系统，以实现明显更高的样本高通量，较低的成本和 像seq鱼中的重复单分子成像的费力进化。它将证明 高空间分辨率（〜5-10μm像素尺寸），高通量 每个操作员）和高含量（全基因组mRNA，蛋白质和非细胞环境）。使用面板 针对细胞外基质（ECM）蛋白的DNA标记抗体的抗体，这种方法进一步允许空间 OMICS测序包括非细胞成分的映射，这些映射在SCRNA-中完全缺少 SEQ或当前空间转录组技术。它非常适合映射人类胶原 包括心脏，主动脉，皮肤和肾脏在内的组织，以提高我们对ECM在正常中的作用的理解 生理，疾病和衰老。我们将追求以下特定目标。在UG3阶段，我们将开发一个 一组新设备，可显着增加组织映射区域（4mmx4mm），发展一个蛋白质组尺度 （〜500种蛋白质共同分析）和包含ECM的空间测序，并发展出新的组织优化 在同一组织载玻片上对HSRTP-Seq进行的协议，并生成一组3D空间转录组 来自人心或主动脉的蛋白质组数据。在UH3阶段，我们将进一步开发多针注射 头以增加样品吞吐量（每天最多100个样品）和映射面积（1.2厘米1.2厘米） 进一步扩展和自动化，开发一种新的组织内模板切换方法来保留完整的组织 在HSRTP-SEQ进行同一组织滑动并构建3D的其他测量之后的截面 组织地图集，并最终开发出优化的PFA和FFPE组织方案，以生成3D多词 来自人类心脏，主动脉，皮肤和肾脏的组织地图集数据（>每个样品的组织切片）。