Visualization of the transcriptome in situ has proven to be a valuable tool in exploring single-cell RNA-sequencing data, providing an additional dimension to investigate spatial cell typing and cell atlases, disease architecture or even data driven discoveries. The field of spatially resolved transcriptomic technologies is emerging as a vital tool to profile gene-expression, continuously pushing current methods to accommodate larger gene panels and larger areas without compromising throughput efficiency. Here, we describe a new version of the in situ sequencing (ISS) method based on padlock probes and rolling circle amplification. Modifications in probe design allows for a new barcoding system via sequence-by-hybridization chemistry for improved spatial detection of RNA transcripts. Due to the amplification of probes, amplicons can be visualized with standard epifluorescence microscopes with high-throughput efficiency and the new sequencing chemistry removes limitations bound by sequence-by-ligation chemistry of ISS. Here we present hybridization-based in situ sequencing (HybISS) that allows for increased flexibility and multiplexing, increased signal-to-noise, all without compromising throughput efficiency of imaging large fields of view. Moreover, the current protocol is demonstrated to work on human brain tissue samples, a source that has proven to be difficult to work with image-based spatial analysis techniques. Overall, HybISS technology works as a target amplification detection method for improved spatial transcriptomic visualization, and importantly, with an ease of implementation.

Hybridization-based In Situ Sequencing (HybISS): spatial transcriptomic detection in human and mouse brain tissue