GSE200481
Spatial Total RNA-Sequencing of regenerating mouse hindlimb muscle and Type 1-Lang reovirus-infected mouse heart
Spatial transcriptomics reveals the spatial context of gene expression, but current methods are limited to assaying endogenously polyadenylated (A-tailed) RNA transcripts. Here we demonstrate that enzymatic in situ polyadenylation of RNAs enables detection of the full spectrum of RNAs, expanding the scope of sequencing-based spatial transcriptomics to the total transcriptome. We apply this Spatial Total RNA-Sequencing (STRS) approach to spatially map noncoding RNAs, newly transcribed RNAs, and non-host RNAs at the tissue-scale in the study skeletal muscle regeneration and viral-induced myocarditis. Our analyses reveal the spatial patterns of noncoding RNA expression with near-cellular resolution, identify noncoding transcripts which exhibit zonal variation in skeletal muscle regeneration, and highlight host transcriptional responses highly associated with local viral RNA abundance. Our in situ polyadenylation strategy relies on a brief, low-cost add-on to a widely used protocol for spatial RNA-sequencing, and thus could be broadly and quickly adopted. Spatial RNA-sequencing of the total transcriptome will enable new insights into spatial gene regulation and biology. Overall design: Spatial total RNA-sequencing was performed using a modified version of the Visium protocol. After dissection, tissues were embedded in OCT compound and frozen fresh in liquid nitrogen. 10um thick tissue sections were mounted onto the Visium Spatial Gene Expression v1 slides. For heart samples, one tissue section was placed into each 6x6mm capture area. For skeletal muscle samples, two tibialis anterior sections were placed into each capture area. After sectioning, tissue sections were fixed in methanol for 20 minutes at -20oC. Next, H&E staining was performed according to the Visium protocol, and tissue sections were imaged. After imaging, the slide was placed into the Visium Slide Cassette. In situ polyadenylation was then performed using yeast Poly(A) Polymerase (Thermo Scientific, cat #74225Z25KU). First, samples were equilibrated by adding 100ul of 1X wash buffer (20ul 5X yPAP Reaction Buffer, 2ul 40U/ul Protector RNase Inhibitor, 78ul nuclease-free H2O) to each capture area and incubating at room temperature for 30 seconds. The buffer was then removed. Next, 75ul of yPAP enzyme mix (15ul 5X yPAP Reaction Buffer, 3ul of 600U/ul yPAP enzyme, 1.5ul 25mM ATP, 3ul 40U/ul Protector RNase Inhibitor, 52.5ul nuclease-free H2O) was added to each reaction chamber. The reaction chambers were then sealed and the slide cassette was incubated at 37oC for 25 minutes. The enzyme mix was then removed. Prior to running STRS, optimal tissue permeabilization time for both heart and skeletal muscle samples was determined to be 15 minutes using the Visium TIssue Optimization Kit from 10x Genomics. Following in situ polyadenylation, the standard Visium library preparation was followed to generate cDNA and final sequencing libraries. The libraries were then pooled and sequenced according to guidelines in the Visium Spatial Gene Expression protocol using either a NextSeq 500 or NextSeq 2000.