Highly multiplexed subcellular RNA sequencing in situ

By Je H. Lee, Evan R. Daugharthy, J. Scheiman, Reza Kalhor, Joyce L. Yang, Thomas C. Ferrante, Richard Terry, Sauveur S. F. Jeanty, Chao Li, Ryoji Amamoto and 9 more. Science, 2014

944 citations recorded by the source corpus.

Transcripts Visualized in Situ Despite advances, current methods for single-cell sequencing are unable to resolve transcript location within the cell, so Lee et al. (p. 1360, published online 27 February) developed a method of fluorescent in situ RNA sequencing (FISSEQ) that works in vivo to show messenger RNA localization within cells. The method amplifies complementary DNA targets by rolling circle amplification, and then in situ cross-linking locks amplicons to produce ample, highly localized templates for three-dimensional sequencing. The technique was tested in fibroblasts to reveal the differences between individual cells during wound repair. Reads of cellular RNA transcripts demonstrate spatial expression differences during simulated wound healing. Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample. Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/1e13215d408eaf4b91ff55c73d786cf98aa21a4f

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Citations in the corpus

Methods and applications for single-cell and spatial multi-omics (2023, background): “Both targeted and untargeted approaches (for example, ISS 186 , ExSeq 125 and FISSEQ 187,188 ) exist for transcript identification, in which highly specific padlock probes are hybridized to in situ-synthesized cDNA sequences, followed by probe ligation and rolling circle amplification (RCA) of…”
Single-cell RNA-seq: advances and future challenges (2014, neutral): “For example, the already discussed in situ sequencing (127) can detect thousands of transcripts while maintaining the natural environment of the cell either from cells grown in a monolayer or a tissue section.”
In situ transcription profiling of single cells reveals spatial organization of cells in the mouse hippocampus (2016, neutral): “In comparison, single cell RNA-seq measurements are 5–20% efficient based on spike-in controls and in situ sequencing is less than 1% efficient (Darmanis et al., 2015; Klein et al., 2015; Lee et al., 2014; Macosko et al., 2015; Tasic et al., 2016; Zeisel et al., 2015; Ståhl et al., 2016).”
RNA-Seq methods for transcriptome analysis (2016, methodology): “Another method, fluorescent in situ RNA sequencing (FISSEQ), combines in situ amplification with sequencing by oligonucleotide ligation.(98) SEQUENCING OF OTHER RNA SPECIES”
Obstacles and opportunities in the functional analysis of extracellular vesicle RNA – an ISEV position paper (2017, methodology): “The recent development of a high throughput method to determine subcellular localisation of RNA in situ [165] may facilitate the identification of such bias in the future.”
The Human Tumor Atlas Network: Charting Tumor Transitions across Space and Time at Single-Cell Resolution (2020, neutral): “Recent advances in single-cell and multiplexed spatial analysis of tissue allow us to interrogate this complexity at unprecedented resolution (Angelo et al., 2014; Buenrostro et al., 2015; Chen et al., 2016a; Chen et al., 2016b; Du et al., 2019; Gaublomme et al., 2019; Gierahn et al., 2017; Giesen e”
Genome-wide RNA Tomography in the zebrafish embryo. (2014, background): “In situ sequencing of RNA in intact tissues has the potential to provide direct information about the spatial organization of the transcriptome (Ke et al., 2013; Lee et al., 2014).”
Single-Cell and Single-Molecule Analysis of Gene Expression Regulation (2016, methodology): “Moreover, fluorescence in situ RNA sequencing (FISSEQ) allows transcriptome sequencing by oligonucleotide ligation and detection (SOLiD) of reverse-transcribed cDNA inside cells (82).”
Exponential scaling of single-cell RNA-seq in the past decade (2017, methodology): “ging (Moffitt et al., 2016a, 2016b; Shah et al., 2016) . Recent promising work has shown untargeted sequencing of cDNA from mRNA inside cells using the fluorescent in situ sequencing method (FISSEQ) (Lee et al., 2014, 2015). The ability to investigate the spatial context of gene expression will be i”
A Single-Cell Transcriptomic Map of the Human and Mouse Pancreas Reveals Inter- and Intra-cell Population Structure (2016, neutral): “Although in situ methods are able to scale up to hundreds or even thousands of genes (Chen et al., 2015; Lee et al., 2014; Lubeck et al., 2014), RNA-seq has the unique advantage that it can detect transcripts comprehensively on a genomic scale.”
Statistical analysis of spatial expression patterns for spatially resolved transcriptomic studies (2019, methodology): “Some of these techniques, such as targeted in situ sequencing (ISS) 8 and FISSEQ 9 , are based on in situ RNA sequencing.”
Computational and analytical challenges in single-cell transcriptomics (2015, background): “If the spatial location of a cell is known 67,68 , in some tissues it can be reasonable to hypothesize that cells located closer to one another are more likely to belong to the same type than more distant cells.”