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44 reviewsSUMMARYPerforming total RNA profiling on formalin-fixed, paraffin-embedded (FFPE) samples, the predominant sample conservation method in clinical practice, remains challenging for current spatial transcriptomics techniques. Here, we introduce Stereo-seq V2, which employs random primers to capture and sequence RNAsin situ on FFPE sections and provides single-cell resolution. The random-priming-based strategy offers unbiased transcript capturing and uniform gene body coverage, which increase the sensitivity to marker genes,the efficiency of non-polyadenylation (poly(A)) RNA profiling, and immune repertoire coverage. We demonstrated the robust performance of Stereo-seq V2 on clinical FFPE samples using triple-negative breastcancer (TNBC) sections and identified tumor-specific alternative splicing events. In a Mycobacterium tuberculosis (Mtb)-infected mouse model, we monitored gene expression dynamics of host and pathogen transcriptomes simultaneously by utilizing Stereo-seq V2. We also assembled immune repertoires and identifiedMtb-specific BCR clones, which could also be observed in human tuberculous lung samples. These resultshighlight Stereo-seq V2’s potential in biomedical research and personalized medicine.
