Highly Multiplexed Single-Cell In Situ RNA and DNA Analysis by Consecutive Hybridization

Knowledge of the copy numbers of transcripts and genomic loci in their natural spatial contexts plays an important role in our understanding of biology and medicine. Fluorescent hybridization probes have emerged as a powerful tool to profile transcripts and genomic loci in single cells in situ. However, the number of different nucleic acid species that can be quantified by fluorescence imaging-based methods is limited. Here, we report a highly multiplexed in situ hybridization approach for spatial transcriptomics and genomics analysis. In this approach, each nucleic acid molecule is visualized as a fluorescent spot at its natural cellular context throughout the consecutive cycles of fluorescence in situ hybridization. In each analysis cycle, fluorescent oligonucleotide probes stain the nucleic acid targets by hybridizing to the probes applied in the previous cycle. And these probes also introduce the binding sites for the next cycle probes. Through reiterative cycles of hybridization, imaging, and photobleaching, unique color sequences are generated as barcodes for varied nucleic acids. With multi-color staining and reiterative cycles, tens of thousands of different transcripts or genomic loci could be precisely profiled in individual cells in situ.