Hybridization-based in situ sequencing (HybISS) for spatially resolved transcriptomics in human and mouse brain tissue

Abstract Visualization of the transcriptome in situ has proven to be a valuable tool in exploring single-cell RNA-sequencing data, providing an additional spatial dimension to investigate multiplexed gene expression, cell types, disease architecture or even data driven discoveries. In situ sequencing (ISS) method based on padlock probes and rolling circle amplification has been used to spatially resolve gene transcripts in tissue sections of various origins. Here, we describe the next iteration of ISS, HybISS, hybridization-based in situ sequencing. Modifications in probe design allows for a new barcoding system via sequence-by-hybridization chemistry for improved spatial detection of RNA transcripts. Due to the amplification of probes, amplicons can be visualized with standard epifluorescence microscopes for high-throughput efficiency and the new sequencing chemistry removes limitations bound by sequence-by-ligation chemistry of ISS. HybISS design allows for increased flexibility and multiplexing, increased signal-to-noise, all without compromising throughput efficiency of imaging large fields of view. Moreover, the current protocol is demonstrated to work on human brain tissue samples, a source that has proven to be difficult to work with image-based spatial analysis techniques. Overall, HybISS technology works as a targeted amplification detection method for improved spatial transcriptomic visualization, and importantly, with an ease of implementation.

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Hybridization-based In Situ Sequencing (HybISS): spatial transcriptomic detection in human and mouse brain tissue

10.1101/2020.02.03.931618 ◽

2020 ◽

Cited By ~ 3

Author(s):

Daniel Gyllborg ◽

Christoffer Mattsson Langseth ◽

Xiaoyan Qian ◽

Sergio Marco Salas ◽

Markus M. Hilscher ◽

...

Keyword(s):

Brain Tissue ◽

Rolling Circle Amplification ◽

Probe Design ◽

Sequencing Data ◽

Human Brain Tissue ◽

Tissue Samples ◽

Rolling Circle ◽

Spatially Resolved ◽

Spatial Detection

Visualization of the transcriptome in situ has proven to be a valuable tool in exploring single-cell RNA-sequencing data, providing an additional dimension to investigate spatial cell typing and cell atlases, disease architecture or even data driven discoveries. The field of spatially resolved transcriptomic technologies is emerging as a vital tool to profile gene-expression, continuously pushing current methods to accommodate larger gene panels and larger areas without compromising throughput efficiency. Here, we describe a new version of the in situ sequencing (ISS) method based on padlock probes and rolling circle amplification. Modifications in probe design allows for a new barcoding system via sequence-by-hybridization chemistry for improved spatial detection of RNA transcripts. Due to the amplification of probes, amplicons can be visualized with standard epifluorescence microscopes with high-throughput efficiency and the new sequencing chemistry removes limitations bound by sequence-by-ligation chemistry of ISS. Here we present hybridization-based in situ sequencing (HybISS) that allows for increased flexibility and multiplexing, increased signal-to-noise, all without compromising throughput efficiency of imaging large fields of view. Moreover, the current protocol is demonstrated to work on human brain tissue samples, a source that has proven to be difficult to work with image-based spatial analysis techniques. Overall, HybISS technology works as a target amplification detection method for improved spatial transcriptomic visualization, and importantly, with an ease of implementation.

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Automated identification of the mouse brain’s spatial compartments from in situ sequencing data

BMC Biology ◽

10.1186/s12915-020-00874-5 ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Gabriele Partel ◽

Markus M. Hilscher ◽

Giorgia Milli ◽

Leslie Solorzano ◽

Anna H. Klemm ◽

...

Keyword(s):

Gene Expression ◽

Mouse Brain ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Cell Types ◽

Sequencing Data ◽

Tissue Samples ◽

Gene Markers ◽

Smooth Transitions

Abstract Background Neuroanatomical compartments of the mouse brain are identified and outlined mainly based on manual annotations of samples using features related to tissue and cellular morphology, taking advantage of publicly available reference atlases. However, this task is challenging since sliced tissue sections are rarely perfectly parallel or angled with respect to sections in the reference atlas and organs from different individuals may vary in size and shape and requires manual annotation. With the advent of in situ sequencing technologies and automated approaches, it is now possible to profile the gene expression of targeted genes inside preserved tissue samples and thus spatially map biological processes across anatomical compartments. Results Here, we show how in situ sequencing data combined with dimensionality reduction and clustering can be used to identify spatial compartments that correspond to known anatomical compartments of the brain. We also visualize gradients in gene expression and sharp as well as smooth transitions between different compartments. We apply our method on mouse brain sections and show that a fully unsupervised approach can computationally define anatomical compartments, which are highly reproducible across individuals, using as few as 18 gene markers. We also show that morphological variation does not always follow gene expression, and different spatial compartments can be defined by various cell types with common morphological features but distinct gene expression profiles. Conclusion We show that spatial gene expression data can be used for unsupervised and unbiased annotations of mouse brain spatial compartments based only on molecular markers, without the need of subjective manual annotations based on tissue and cell morphology or matching reference atlases.

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Toehold-initiated Rolling Circle Amplification for Visualizing Individual MicroRNAs In Situ in Single Cells

Angewandte Chemie ◽

10.1002/ange.201309388 ◽

2014 ◽

Vol 126 (9) ◽

pp. 2421-2425 ◽

Cited By ~ 49

Author(s):

Ruijie Deng ◽

Longhua Tang ◽

Qianqian Tian ◽

Ying Wang ◽

Lei Lin ◽

...

Keyword(s):

Single Cells ◽

Rolling Circle Amplification ◽

Rolling Circle

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A novel coplanar probe design for fast scanning of edema in human brain tissue via dielectric measurements

Sensors and Actuators B Chemical ◽

10.1016/j.snb.2015.06.002 ◽

2015 ◽

Vol 220 ◽

pp. 522-527 ◽

Cited By ~ 5

Author(s):

T. Reinecke ◽

L. Hagemeier ◽

S. Ahrens ◽

Y. Doroschenko ◽

M. Klintschar ◽

...

Keyword(s):

Human Brain ◽

Brain Tissue ◽

Probe Design ◽

Dielectric Measurements ◽

Human Brain Tissue

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Rolling Circle Amplification with Padlock Probes for In Situ Detection of RNA Analytes

Rolling Circle Amplification (RCA) ◽

10.1007/978-3-319-42226-8_9 ◽

2016 ◽

pp. 99-105

Author(s):

Anja Mezger ◽

Malte Kühnemund ◽

Mats Nilsson

Keyword(s):

Rolling Circle Amplification ◽

Rolling Circle ◽

Padlock Probes ◽

In Situ Detection

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In Situ Detection of Anaplasma spp. by DNA Target-Primed Rolling-Circle Amplification of a Padlock Probe and Intracellular Colocalization with Immunofluorescently Labeled Host Cell von Willebrand Factor

Journal of Clinical Microbiology ◽

10.1128/jcm.02197-07 ◽

2008 ◽

Vol 46 (7) ◽

pp. 2314-2319 ◽

Cited By ~ 15

Author(s):

H. L. Wamsley ◽

A. F. Barbet

Keyword(s):

Host Cell ◽

Von Willebrand Factor ◽

Rolling Circle Amplification ◽

Padlock Probe ◽

Rolling Circle ◽

In Situ Detection ◽

Von Willebrand ◽

Willebrand Factor

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Visualization and Enumeration of Bacteria Carrying a Specific Gene Sequence by In Situ Rolling Circle Amplification

Applied and Environmental Microbiology ◽

10.1128/aem.71.12.7933-7940.2005 ◽

2005 ◽

Vol 71 (12) ◽

pp. 7933-7940 ◽

Cited By ~ 29

Author(s):

Fumito Maruyama ◽

Takehiko Kenzaka ◽

Nobuyasu Yamaguchi ◽

Katsuji Tani ◽

Masao Nasu

Keyword(s):

Gene Sequence ◽

Rolling Circle Amplification ◽

Specific Gene ◽

Toxin Gene ◽

Dna Uptake ◽

Fluorescent Intensity ◽

Prolonged Incubation ◽

Rolling Circle ◽

E Coli

ABSTRACT Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count single Escherichia coli cells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes inside E. coli cells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA. E. coli cells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 106-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria.

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