In situ transcriptome profiling in single cells

摘要 我们最近开发了内含子seqFISH(顺序荧光原位杂交)来多重10,421 基因直接存在于单个细胞中。我们发现10,421基因的新生转录组图谱可以识别细胞 类型以及捕捉细胞的轨迹。我们进一步证明了我们可以执行mRNA 在10,421基因内含子seqFISH检测后，在相同的细胞中进行免疫染色。 我们建议开发这项技术，作为单细胞RNAseq的潜在替代方法 HuBMAP可直接在组织中原位鉴定细胞类型。特别是，我们将熟练地进行原位扩增 内含子序列的杂交链式反应(HCR)等方法。我们之前已经证明了mRNA 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 并实现对seqFISH条形码的稳健解码。我们将验证整合的内含子和mRNA序列 该项目的UG3阶段在小鼠海马体中的协议。同样在UG3阶段，我们将开发 整合内含子序列和信使核糖核酸序列以及单细胞RNAseq数据的计算工具。 在UH3阶段，我们将把这项技术转化为人体组织，重点是人类乳房组织 由希沃特博士在希望之城提供。我们还将与HuBMAP中的组织映射中心合作 加速将这项技术转化为多种组织类型的计划。在UH3阶段，我们将 生成包含内含子图谱、信使核糖核酸图谱和蛋白质的百万个人体组织细胞空间图谱 每一个细胞中的丰度。我们将进一步开发计算工具来分析空间丰富的 基因在组织中，并使用新生转录组产生假时间的发育轨迹 数据。综上所述，我们将开发一个高通量的基于原位成像的细胞表征平台 使用内含子和信使核糖核酸序列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.