In situ transcriptome profiling in single cells

总结 我们最近开发了内含子seqFISH（顺序荧光原位杂交）， 基因直接在单个细胞中。我们发现，10，421个基因的新生转录组谱可以识别细胞， 类型以及捕捉细胞的轨迹。我们进一步证明，我们可以执行mRNA seqFISH以及在10，421个基因内含子seqFISH测量后在相同细胞中的免疫染色。 我们建议开发这种技术，作为单细胞RNAseq的潜在替代方法， HuBMAP直接在组织中原位表征细胞类型。特别是，我们将擅长原位扩增， 方法如杂交链反应（HCR）到内含子seqFISH。我们之前已经证明， 具有HCR扩增的seqFISH在克服自体荧光背景方面在组织中表现异常 并且能够对seqFISH条形码进行鲁棒解码。我们将验证整合的内含子和mRNA seqFISH 在UG3阶段的项目中，在小鼠海马体中进行了实验。此外，在UG3阶段，我们将开发 计算工具来整合内含子seqFISH数据与mRNA seqFISH以及单细胞RNAseq数据。 在UH3阶段，我们将把这项技术应用于人体组织，重点是人体乳腺组织 由City of Hope的Seewaldt医生提供我们还将与HuBMAP中的组织映射中心合作 该计划旨在加速将该技术转化为许多组织类型。在UH3阶段，我们将 生成包含内含子谱、mRNA谱和蛋白质的人体组织的百万细胞空间图谱 每一个细胞中的丰度。我们将进一步开发计算工具，以分析空间富集 基因在组织中，并使用新生转录组产生发育轨迹的伪时间 数据综上所述，我们将开发一个高通量的原位成像平台来表征细胞 使用内含子和mRNA seqFISH技术的细胞类型和未来轨迹。

Summary We have recently developed intron seqFISH (sequential Fluorescence in situ hybridization) to multiplex 10,421 genes directly in single cells. We showed that the 10,421 gene nascent transcriptome profile can identify cell types as well as capture the trajectory of the cells. We further demonstrated that we can perform mRNA seqFISH as well as immunostaining in the same cells following the 10,421 gene intron seqFISH measurement. We propose to develop this technology as a potential alternative approach to single cell RNAseq for the HuBMAP to characterize cell types directly in situ in tissues. In particular, we will adept in situ amplification methods such as hybridization chain reaction (HCR) to intron seqFISH. We had previously shown that mRNA seqFISH with HCR amplification performs exceptionally in tissues in overcoming autofluorescence background and enable robust decoding seqFISH barcodes. We will validate the integrated intron and mRNA seqFISH protocol in the mouse hippocampus in the UG3 phase of the project. Also in UG3 phase, we will develop computational tools to integrate intron seqFISH data with mRNA seqFISH as well as single cell RNAseq data. In the UH3 phase, we will translate the technology to human tissues, with a focus on human mammary tissues provided by Dr. Seewaldt at City of Hope. We will also work with the tissue mapping centers in the HuBMAP program to accelerate the translation of this technology to many tissue types. In the UH3 phase, we will generate million cell spatial atlas of human tissues containing intron profiles, mRNA profiles and protein abundances in each single cell. We will further develop computational tools to analyze for spatial enrichment of genes in the tissue and generate a pseudotime of developmental trajectories using the nascent transcriptome data. Taken together, we will develop a high throughput in situ imaging based platform to characterize cell types and future trajectories of cells using intron and mRNA seqFISH technologies.