Long Cai’s lab has recently developed intron seqFISH (sequential Fluorescence in situ hybridization) to multiplex 10,421 genes directly in single cells. They showed that the 10,421 gene nascent transcriptome profile can identify cell types as well as capture the trajectory of the cells, and further demonstrated that they can perform mRNA seqFISH as well as immunostaining in the same cells following the 10,421 gene intron seqFISH measurement.

For their project, this team is developing this technology as a potential alternative approach to single cell RNAseq for HuBMAP to characterize cell types directly in situ in tissues. In particular, they adapt in situ amplification methods such as hybridization chain reaction (HCR) to intron seqFISH. They have previously shown that mRNA seqFISH with HCR amplification performs exceptionally in tissues in overcoming autofluorescence background and enables robust decoding of seqFISH barcodes. They will validate the integrated intron and mRNA seqFISH protocol in the mouse hippocampus in the UG3 phase of the project. Also in the UG3 phase, they will develop computational tools to integrate intron seqFISH data with mRNA seqFISH as well as single cell RNAseq data. In the UH3 phase, they will translate the technology to human tissues, with a focus on human mammary tissues provided by Dr. Seewaldt at City of Hope.

They 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, they will generate a million cell spatial atlas of human tissues containing intron profiles, mRNA profiles and protein abundances in each single cell. They will further develop computational tools to analyze for spatial enrichment of genes in the tissue and generate a pseudo time of developmental trajectories using the nascent transcriptome data. Taken together, they 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.