Scalable in situ single-cell profiling by electrophoretic capture of mRNA

Abstract

Methods to spatially profile the transcriptome are dominated by a trade-off between resolution and throughput. Here, we developed a method named EEL FISH that can rapidly process large tissue samples without compromising spatial resolution. By electrophoretically transferring RNA from a tissue section onto a capture surface, EEL speeds up data acquisition by reducing the amount of imaging needed, while ensuring that RNA molecules move straight down towards the surface, preserving single-cell resolution. We applied EEL on eight entire sagittal sections of the mouse brain and measured the expression patterns of up to 440 genes to reveal complex tissue organisation. Moreover, EEL enabled the study of challenging human samples by removing autofluorescent lipofuscin, so that we could study the spatial transcriptome of the human visual cortex. We provide full hardware specification, all protocols and complete software for instrument control, image processing, data analysis and visualization.

Spatial Atlas of the Mouse Central Nervous System at Molecular Resolution

“…2e). As another example, we confirmed region-specific patterns of astrocyte subtypes 1,37 in the telencephalon (AC_2-3, Mfge8 + ), non-telencephalon (AC_1, Agt + ), cerebellar cortex (AC_4, Gdf10 + ), and fiber tracts (AC_5, Gfap + Mbp + ; AC_6, Gfap + Myoc + ) (Fig. 2e, Extended Data Fig.…”

Single‐Nucleus RNA Sequencing and Spatial Transcriptomics Reveal the Immunological Microenvironment of Cervical Squamous Cell Carcinoma

“…It is worth noting that most spatial transcriptomic techniques capture RNA from permeabilized tissue overlaid on top of a chip in a liquid system, where the lateral diffusion of RNA is unavoidable. [ 38 , 41 , 42 ] This phenomenon is most obvious in cavities within tissues and tissue areas with extremely low RNA abundance, because the capture probes underneath may be far from saturated. Therefore, we combined pathological assessment and marker gene expression patterns (Figure S1 C,D, Supporting Information) to ensure that no obvious lateral diffusion occurred in the stereo chips, and this served as a quality control step.…”

Spatial omics technologies at multimodal and single cell/subcellular level

“…To overcome potential errors and dropouts, most probe-based methods utilize extra hybridization steps added for every few cycles to ensure the rest of the transcripts are detected properly. Current technologies include multiplexed error robust fluorescence in situ hybridization (MERFISH) [25], molecular cartography [26], sequential fluorescence in situ hybridization (seqFISH +) [24], sequent ouroboros single-molecule FISH (osmFISH) [27], Split-FISH [28], hybridization-based in situ sequencing (HybISS) [29], Esper [30,31], CosMx [32], Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA) [33], and combinatorial padlock-probe-amplified FISH (coppaFISH) [34]. Each technology utilizes slightly different probe length, design methods, and protocols.…”