Mapping the total transcriptome at near cellular resolution

Spatial transcriptomics provides context to gene expression information, however most methods are only sensitive to a subset of the RNA species. Many noncoding and microbial RNA molecules lack the poly(A) tail which is used to capture RNAs in sequencing-based methods. These transcripts represent valuable analytes for studying gene regulation and host-microbe interactions, respectively. We and others recently showed that in situ polyadenylation combined with poly(dT) capture enables broad analysis of coding, noncoding, and non-host RNAs. However, current implementations neither attain single-cell resolution or retain spatial information. Recent commercialization of spatial transcriptomics tools have enabled broad adoption of these technologies. Several new tools have been developed which can attain near cellular resolution, including SlideSeq (Seeker, Takara/Curio Bioscience) and StereoSeq (STOmics, BGI). Here we adapt in situ polyadenylation to two commerical spatial transcriptomics platforms with near cellular resolution and show the spatial heterogeneity of the total transcriptome in the infected murine heart and testis. Overall design: Frozen testis from DBA/2J mice and frozen reovirus-induced myocarditic hearts collected at 7 days post infection (dpi) following oral gavage with Reovirus type 1 Lang (T1L) were processed for Spatial Total RNA Sequencing using a modified Stereoseq (STOmics) workflow. Tissues were cryosectioned at 10 µm thickness and mounted onto a 1 cm × 1 cm STOmics Stereoseq chip. Sections were fixed in ice-cold methanol for 30 min at -20°C, followed by staining and imaging according to the manufacturer's Stereoseq/STOmics protocol. In situ polyadenylation was then performed using yeast poly(A) polymerase (yPAP; Thermo Scientific): capture areas were equilibrated with 1× wash buffer, incubated with yPAP enzyme mix at 37°C, and the enzyme was removed by washing. A 15-min enzymatic permeabilization step was subsequently performed prior to continuing with the standard STOmics library preparation workflow to generate cDNA and sequencing libraries.