Probes for INS

Spatial transcriptomics enables the study of localization-indexed gene expression activity in tissues, providing the transcriptional landscape that in turn indicates the potential regulatory networks of gene expression. In situ sequencing (ISS) is a targeted spatial transcriptomic technique, based on padlock probe and rolling circle amplification combined with next-generation sequencing chemistry, for highly multiplexed in situ gene expression profiling. Here, we present improved in situ sequencing (IISS) that exploits a new probing and barcoding approach, combined with advanced image analysis pipelines for high-resolution targeted spatial gene expression profiling. We develop an improved combinatorial probe anchor ligation chemistry using a 2-base encoding strategy for barcode interrogation. The new encoding strategy results in higher signal intensity as well as improved specificity for in situ sequencing, while maintaining a streamlined analysis pipeline for targeted spatial transcriptomics. We show that IISS can be applied to both fresh frozen tissue and formalin-fixed paraffin-embedded tissue sections for single-cell level spatial gene expression analysis, based on which the developmental trajectory and cell-cell communication networks can also be constructed.

The last 5 y have seen the development and widespread adoption of high-plex spatial transcriptomic technology. This technique detects and quantifies mRNA transcripts in situ, meaning that transcriptomic signatures can be sampled from specific cells, structures, lesions, or anatomical regions while conserving the physical relationships that exist within complex tissues. These methods now frequently implement next-generation sequencing, enabling the simultaneous measurement of many targets, up to and including the whole mRNA transcriptome. To date, spatial transcriptomics has been foremost used in the fields of neuroscience and oncology, but there is potential for its use in transplantation sciences. Transplantation has a clear dependence on biopsies for diagnosis, monitoring, and research. Transplant patients represent a unique cohort with multiple organs of interest, clinical courses, demographics, and immunosuppressive regimens. Obtaining high complexity data on the disease processes underlying rejection, tolerance, infection, malignancy, and injury could identify new opportunities for therapeutic intervention and biomarker identification. In this review, we discuss currently available spatial transcriptomic technologies and how they can be applied to transplantation.

The ability to explore life kingdoms is largely drive by innovations and breakthroughs in technology, from the invention of the microscope 350 years ago to the recent emergence of single cell sequencing, by which the scientific community has been able to visualize life at an unprecedented resolution. Most recently, the Spatially Resolved Transcriptomics (SRT) technologies have filled the gap in probing the spatial or even three-dimensional (3D) organization of the molecular foundation behind the molecular mysteries of life, including the origin of different cellular populations developed from totipotent cells and human diseases. In this review, we introduce recent progress and challenges on SRT from the perspectives of technologies and bioinformatic tools, as well as the representative SRT applications. With the currently fast-moving progress of the SRT technologies and promising results from early adopted research projects, we can foresee the bright future of such new tools in understanding life at the most profound analytical level.