Highly Multiplexed Subcellular RNA Sequencing in Situ

Abstract

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 cDNA amplicons are sequenced within a biological sample. Using 30-base reads from 8,742 genes in situ, we examined RNA expression and localization in human primary fibroblasts using 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.

Stochastic electrotransport selectively enhances the transport of highly electromobile molecules

“…For instance, it can take weeks for antibodies to diffuse a few millimeters into fixed tissues (2). The slow nature of diffusive transport has long limited the application of many existing and emerging techniques in biology and medicine to small or thin tissue samples (3)(4)(5)(6)(7).…”

“…However, with the development of in situ molecular interrogation methods (6,13,14) and tissue clearing techniques (2,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) and an emphasis on studying organ-scale tissue as a whole, a pressing need has arisen for a means of expediting the transportation of various molecules into intact tissues. For example, many emerging tissue clearing techniques use surfactant micelles to directly remove lipids from a tissue and thus eliminate light-scattering boundaries to improve optical penetration for holistic visualization (2,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25), but transporting these micelles into the intact tissue via diffusion can take weeks (2,15).…”

“…Diverse methods of tissue labeling are used in many areas of biological research and medical diagnosis for visualizing various biomolecules of interest. However, these techniques have been mostly confined to small samples owing to the difficulty of labeling and examining deep structures in large-scale intact tissues (6,(27)(28)(29)(30)(31). CLARITY and other emerging tissue-clearing techniques (2,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) render intact tissues optically transparent and macromolecule-permeable, allowing examination deep inside Significance Many chemical and biomedical techniques rely on slow diffusive transport because existing pressure-based methods or electrokinetic methods can incidentally damage the sample.…”

State of the art technologies to explore long non‐coding RNAs in cancer

“…However, the technique needs to be refined regarding its read length, sequencing depth and coverage and library preparation 125.…”

Computer vision for image-based transcriptomics

“…Alternatively, transcripts can be visualized indirectly via reverse transcription to cDNA that can be sequenced in situ by padlock probes [13] or oligonucleotide ligation and detection [14,15].…”