Shedding new light on single cell transcriptomics

In recent years, the ability to combine image-based and molecular characterization of samples has become a focus area for better understanding biology in both health and disease. Traditional methods for studying the expression of RNA transcripts, collectively known as transcriptomics (e.g. RNA sequencing), provide a wealth of information about the gene expression profiles of cells but lack the spatial context necessary to appreciate how these patterns contribute to tissue function and organization. Spatial transcriptomics bridges this gap by providing a means to map gene expression patterns in their native tissue context.

The authors of this paper have developed a novel spatial transcriptomic technique called Light-Seq, which uses light to create and read microscopic barcodes that can identify and map RNA in cells and tissues. Existing commercial options often require custom-built and expensive equipment, include high operating costs, and don’t allow for selecting regions or cells of interest prior to experiments. In the Light-Seq process, reverse transcription is first performed to generate cDNA from the sample. Next, a special DNA probe that undergoes rapid photocrosslinking is applied to the sample, and by precisely controlling the spatial and temporal pattern of light exposure, researchers can barcode the cDNA from regions of interests at the single cell level. Unlike existing companies such as Visium, which retroactively maps RNA expression back onto samples, Light-Seq allows visualization of RNA expression even prior to removal of transcripts targeted for sequencing. After the barcoded DNA probes have successfully bound to their targets, the barcodes are amplified through a process called polymerase chain reaction (PCR) and read by a sequencing process. This allows scientists to create a detailed map of RNA expression levels and distribution within the cells and tissues.

Figure 1. Light-Seq leverages unique patterns of light to select regions or cells for barcoding. Once barcoded, cells from selected areas can be genetically profiled using existing sequencing techniques. Subsequently, the same tissue or sample can be used for additional assays such as immunofluorescent staining.

Spatial transcriptomic techniques today have unique drawbacks that range from tissue damage to high operating costs. Light-Seq addresses multiple caveats in the space, does not rely on custom equipment, is significantly more cost-effective than comparable methods, and allows for unbiased deep sequencing at the single cell level. To demonstrate the strengths and capabilities of their method, the authors performed Light-Seq on dopaminergic amacrine cells in the retina, of which only 4 to 8 cells can be found per section. They were able to pre-process the tissue by labeling these cells with a fluorescent marker, select those specific cells for deep sequencing based on the marker, and validate the resulting hits with RNAscope in the same cells in the same tissue. They also demonstrated that the expression profiles collected with Light-Seq were consistent with other established single-cell expression methods. For example, the team found a 98.6% correlation between the retinal layer gene expression data from a published Drop-Seq dataset and that of their own Light-Seq results.  

In summary, Light-Seq is a cutting-edge technique that leverages light to create and read microscopic barcodes for providing spatial transcriptomic data. This method provides an unprecedented approach for unbiased characterization of specific cells and cell types that can be selected for at the single cell level by visual criteria such as fluorescence and morphology.

Edited by Emma Costa.

References

Kishi, J.Y., Liu, N., West, E.R. et al. Light-Seq: light-directed in situ barcoding of biomolecules in fixed cells and tissues for spatially indexed sequencing. Nat Methods 19, 1393–1402 (2022). https://doi.org/10.1038/s41592-022-01604-1