Spatial mapping of the total transcriptome by in situ polyadenylation

Abstract

Spatial transcriptomics reveals the spatial context of gene expression, but current methods are limited to assaying polyadenylated (A-tailed) RNA transcripts. Here we demonstrate that enzymatic in situ polyadenylation of RNA enables detection of the full spectrum of RNAs, expanding the scope of sequencing-based spatial transcriptomics to the total transcriptome. We demonstrate that our spatial total RNA-sequencing (STRS) approach captures coding RNAs, noncoding RNAs and viral RNAs. We apply STRS to study skeletal muscle regeneration and viral-induced myocarditis. Our analyses reveal the spatial patterns of noncoding RNA expression with near-cellular resolution, identify spatially defined expression of noncoding transcripts in skeletal muscle regeneration and highlight host transcriptional responses associated with local viral RNA abundance. STRS requires adding only one step to the widely used Visium spatial total RNA-sequencing protocol from 10x Genomics, and thus could be easily adopted to enable new insights into spatial gene regulation and biology.

Spatial Transcriptomics: Emerging Technologies in Tissue Gene Expression Profiling

“…Recently, ST was expanded to include a poly-adenylation of the 3'-end of all the RNA content, allowing for whole transcriptomics analysis, not limited to mRNA 52 . ST has been widely utilized in various biological studies, including architecture, development, and human diseases 53,54 , however, the main limitation is that it does not resolve spatial transcriptomics at the single-cell resolution.…”

Spatial omics technologies at multimodal and single cell/subcellular level

“…Because the resolution can be limited by the size of the amplicon hybridized on the slide for sequencing, different strategies to improve the UMI and quality of the reads are currently being explored in these technologies. Also, methodologies to capture total transcriptome (viral, coding, and noncoding RNAs) are also developed, such as spatial total RNA-sequencing (STRS) [50].…”

The myonuclear domain in adult skeletal muscle fibres: past, present and future

“…RNA imaging techniques, on the other hand, reveal high‐resolution spatial information but have generally only been applied to a limited number of transcripts. Future work should aim to bring cutting‐edge spatial transcriptomics approaches in muscle systems (McKellar et al., 2021, 2022) to the context of exercise and the myonuclear domain. Adapting modern approaches to resolving the spatial localisation patterns of the full transcriptome in muscle promises to clarify the relationship between myonuclear domains and tissue organisation and provide the statistical power to evaluate mechanisms underlying localisation specificity.…”

“…Collectively, transcriptional compartmentalisation by myonuclei in muscle fibres at rest and during remodelling could have consequences for myonuclear domain regulation. Combining smnRNA‐seq with emerging techniques such as RNA‐scope (Kann & Krauss, 2019) and spatial transcriptomics in muscle (McKellar et al., 2021, 2022; Young et al., 2022) will further define myonuclear subpopulations and their influence on myonuclear specialisation and compartmentalisation. Future work should focus on understanding the mechanisms that can establish and maintain specialised domains within a shared syncytia.…”

“…Most information on the myonuclear domain has related to the production/localisation of mRNA and the protein from that message; essentially nothing is known about non‐coding RNA in this context. Recent advances in spatial transcriptomic profiling may illuminate how non‐coding and ribosomal RNA behave with respect to myonuclear domains (McKellar et al., 2022). Advances in spatial proteomics will further augment these efforts (Lundberg & Borner, 2019; Mund et al., 2022).…”