Incorporating single molecule in situ hybridization approach to single cell sequencing workflows [thesis]

Xiaoting Zhang, Rafael Kramann, Ivan G. Costa
2022
Renal fibrosis is considered as a key therapeutic target of CKD, however, no approved specifically targeted to renal fibrosis therapies exist currently. This is because the origins of scar-producing myofibroblasts remain controversial. Single-cell RNA-seq(scRNA-seq), which measures global gene expression, have already been avidly adopted by the fibrosis research community to comprehensively profile the cellular and molecular that drives lung and liver fibrosis. However, scRNA-seq data lack of
more » ... atial information, which make single molecule in situ hybridization(smFISH) an important complement to single cell data. But approaches for spatially resolved, multiplexed (more than three) gene expression profiling on complex tissues are lacking. Objective: Incorporating fully developed RNAscope FISH approach to scRNA-seq workflow to decoding the origins of myofibroblasts and developed a multiplexing (up to six) smFISH (SABER FISH) protocol in kidney tissue and in-vitro cells. Methods: Based on our scRNA-seq data from our working group which profile the transcriptomes and map of all matrix-producing cells. Fully developed RNAscope FISH approach to validate the single cell results and elucidate the context-dependent transcriptional regulation during development. Developed SABER FISH in mouse kidney tissue and in-vitro cells according to the published protocol. Results: Distinct pericyte and fibroblast subpopulations are the major source of myofibroblasts in human kidney fibrosis. RNAscope FISH is a powerful technique for measuring both RNA abundance and localization with single cell resolution in tissue context. SABER FISH protocol had been successfully developed in mouse kidney tissue and in-vitro cells. It is cost effective and reliable smFISH workflow, which could visualize more than three different RNAs in the same staining by using orthogonal chemistries. Conclusion: smFISH technology can interrogate gene expression with single cell resolution in complex tissue context and cultured cells, which is a useful [...]
doi:10.18154/rwth-2022-03705 fatcat:4npgg2dnwbg3rmc7ywhaiz2biy