Various approaches for spatially resolved transcriptomics have been devised, each with their own trade-offs. The detection of transcripts in situ directly on tissue slides provides high, subcellular spatial resolution but is limited in its transcriptomic comprehensiveness: detection based on probe hybridization using single-molecule fluorescence in situ hybridization (smFISH) is limited by the number of distinct fluorescent probes, and in situ RNA-seq is limited by visual crowding of transcripts. Alternative methods extract small tissue patches (such as by laser-capture microdissection), incorporate separate molecular barcodes during library preparation as a tag for the location of each source patch, then carry out standard RNA-seq in solution. These alternative approaches only interrogate the small extracted patches, and each sample must be individually tagged.
Ståhl, Salmén et al. sought to combine some of the strongest features of both types of approach. They labelled glass slides with ∼200 million reverse transcription oligonucleotide probe molecules: ∼1,000 clusters of probes were positioned 200 μm apart in a grid, and each cluster included a different spatial barcode within the probes. Initial tests using tissue sections of the mouse olfactory bulb showed that the section could first be imaged by regular haematoxylin and eosin (H&E) staining on the slide, then the tissue could be lysed, the RNA captured on the slide and converted to cDNA with minimal lateral diffusion. The cDNA was then pooled and subjected to RNA-seq in solution. The spatial barcodes allowed the reads to be retrospectively linked to their original location on the slide.
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