scholarly journals Probe-Seq enables transcriptional profiling of specific cell types from heterogeneous tissue by RNA-based isolation

2019 ◽  
Author(s):  
Ryoji Amamoto ◽  
Mauricio D. Garcia ◽  
Emma R. West ◽  
Jiho Choi ◽  
Sylvain W. Lapan ◽  
...  
Keyword(s):  
Transcriptional Profiling ◽  
Cell Types ◽  
Cell Populations ◽  
Specific Cell ◽  
Rna Profiling ◽  
Complementary Method ◽  
Dissociated Cells ◽  
Multiple Cell ◽  
Heterogeneous Tissue

ABSTRACTRecent transcriptional profiling technologies are uncovering previously-undefined cell populations and molecular markers at an unprecedented pace. While single cell RNA (scRNA) sequencing is an attractive approach for unbiased transcriptional profiling of all cell types, a complementary method to isolate and sequence specific cell populations from heterogeneous tissue remains challenging. Here, we developed Probe-Seq, which allows deep transcriptional profiling of specific cell types isolated using RNA as the defining feature. Dissociated cells are labelled using fluorescent in situ hybridization (FISH) for RNA, and then isolated by fluorescent activated cell sorting (FACS). We used Probe-Seq to purify and profile specific cell types from mouse, human, and chick retinas, as well as the Drosophila midgut. Probe-Seq is compatible with frozen nuclei, making cell types within archival tissue immediately accessible. As it can be multiplexed, combinations of markers can be used to create specificity. Multiplexing also allows for the isolation of multiple cell types from one cell preparation. Probe-Seq should enable RNA profiling of specific cell types from any organism.

scholarly journals Probe-Seq enables transcriptional profiling of specific cell types from heterogeneous tissue by RNA-based isolation

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Ryoji Amamoto ◽  
Mauricio D Garcia ◽  
Emma R West ◽  
Jiho Choi ◽  
Sylvain W Lapan ◽  
...  
Keyword(s):  
Transcriptional Profiling ◽  
Cell Types ◽  
Cell Populations ◽  
Specific Cell ◽  
Rna Profiling ◽  
Complementary Method ◽  
Dissociated Cells ◽  
Multiple Cell ◽  
Heterogeneous Tissue

Recent transcriptional profiling technologies are uncovering previously-undefined cell populations and molecular markers at an unprecedented pace. While single cell RNA (scRNA) sequencing is an attractive approach for unbiased transcriptional profiling of all cell types, a complementary method to isolate and sequence specific cell populations from heterogeneous tissue remains challenging. Here, we developed Probe-Seq, which allows deep transcriptional profiling of specific cell types isolated using RNA as the defining feature. Dissociated cells are labeled using fluorescent in situ hybridization (FISH) for RNA, and then isolated by fluorescent activated cell sorting (FACS). We used Probe-Seq to purify and profile specific cell types from mouse, human, and chick retinas, as well as from Drosophila midguts. Probe-Seq is compatible with frozen nuclei, making cell types within archival tissue immediately accessible. As it can be multiplexed, combinations of markers can be used to create specificity. Multiplexing also allows for the isolation of multiple cell types from one cell preparation. Probe-Seq should enable RNA profiling of specific cell types from any organism.

scholarly journals Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems

2020 ◽  
Author(s):  
Shahar Alon ◽  
Daniel R Goodwin ◽  
Anubhav Sinha ◽  
Asmamaw T Wassie ◽  
Fei Chen ◽  
...  
Keyword(s):  
Splice Variants ◽  
Metastatic Breast ◽  
Cell Types ◽  
Specific Cell ◽  
Long Read ◽  
Multiple Cell ◽  
Cancer Biopsy ◽  
Subcellular Resolution ◽  
Mouse Visual Cortex

Abstract:Methods for highly multiplexed RNA imaging are limited in spatial resolution, and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to mouse brain, yielding readout of thousands of genes, including splice variants and novel transcripts. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in neurons of the mouse hippocampus, revealing patterns across multiple cell types; layer-specific cell types across mouse visual cortex; and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus ExSeq enables highly multiplexed mapping of RNAs, from nanoscale to system scale.One Sentence SummaryIn situ sequencing of physically expanded specimens enables multiplexed mapping of RNAs at nanoscale, subcellular resolution.

scholarly journals FIN-Seq: transcriptional profiling of specific cell types from frozen archived tissue of the human central nervous system

2019 ◽  
Author(s):  
Ryoji Amamoto ◽  
Emanuela Zuccaro ◽  
Nathan C Curry ◽  
Sonia Khurana ◽  
Hsu-Hsin Chen ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Rna Sequencing ◽  
Human Tissue ◽  
Transcriptional Profiling ◽  
Cell Types ◽  
Specific Cell ◽  
Human Central Nervous System ◽  
Tissue Samples ◽  
Rna Profiling

Abstract Thousands of frozen, archived tissue samples from the human central nervous system (CNS) are currently available in brain banks. As recent developments in RNA sequencing technologies are beginning to elucidate the cellular diversity present within the human CNS, it is becoming clear that an understanding of this diversity would greatly benefit from deeper transcriptional analyses. Single cell and single nucleus RNA profiling provide one avenue to decipher this heterogeneity. An alternative, complementary approach is to profile isolated, pre-defined cell types and use methods that can be applied to many archived human tissue samples that have been stored long-term. Here, we developed FIN-Seq (Frozen Immunolabeled Nuclei Sequencing), a method that accomplishes these goals. FIN-Seq uses immunohistochemical isolation of nuclei of specific cell types from frozen human tissue, followed by bulk RNA-Sequencing. We applied this method to frozen postmortem samples of human cerebral cortex and retina and were able to identify transcripts, including low abundance transcripts, in specific cell types.

Laser Capture Microdissection: Understanding the Techniques and Implications for Molecular Biology in Nursing Research Through Analysis of Breast Cancer Tumor Samples

2011 ◽  
Vol 13 (3) ◽  
pp. 297-305 ◽  
Author(s):  
Karen L. Zanni ◽  
Garrett K. Chan
Keyword(s):  
Laser Capture Microdissection ◽  
Breast Biopsy ◽  
Ductal Carcinoma ◽  
Pcr Amplification ◽  
Cell Types ◽  
Cell Populations ◽  
Specific Cell ◽  
Pure Cell ◽  
Heterogeneous Tissue ◽  
Laser Capture

Aim: The purpose of this paper is to review the techniques and implications of laser capture microdissection (LCM) to isolate tissue and DNA of interest using breast biopsy tissue as an example. Background: Tissues are a heterogeneous mix of different cell types, and molecular alterations are often specific to a single cell type. An accurate correlation of molecular and morphologic pathologies requires the ability to procure pure populations of morphologically similar cells for molecular analysis. LCM is a technique for isolating highly pure cell populations of morphologically similar cells from a heterogeneous tissue section. Method: Nine invasive, paraffin-embedded breast biopsy specimens were obtained and analyzed. Depending on the size of the lesion, 500—1,000 shots using the 7.5- or 15-µm infrared laser beam were utilized to obtain an average of 2,000 cells. DNA was isolated from normal tissue and carcinomas and polymerase chain reaction (PCR) amplification was examined by agarose gel electrophoresis. The HER2/neu gene was amplified by standard PCR. A second round of PCR using nested primers to re-amplify the HER2/neu fragment was performed. Results: Amplification of the HER2/neu gene with DNA isolated from pure cell populations by LCM was performed. The results indicated that 22% of the cases studied were positive for HER2/neu amplifications, which corresponds to the literature regarding HER2/neu amplification/overexpression. HER2/neu amplification could be detected as early as the ductal carcinoma in situ (DCIS) stage. Conclusion: LCM is an accurate and reliable method to acquire nucleic acid and protein profiles from a specific cell population in heterogeneous tissue.

scholarly journals Expansion sequencing: Spatially precise in situ transcriptomics in intact biological systems

Science ◽  
2021 ◽  
Vol 371 (6528) ◽  
pp. eaax2656 ◽  
Author(s):  
Shahar Alon ◽  
Daniel R. Goodwin ◽  
Anubhav Sinha ◽  
Asmamaw T. Wassie ◽  
Fei Chen ◽  
...  
Keyword(s):  
Splice Variants ◽  
Metastatic Breast ◽  
Cell Types ◽  
Specific Cell ◽  
Mouse Hippocampus ◽  
Long Read ◽  
Multiple Cell ◽  
Cancer Biopsy ◽  
Mouse Visual Cortex

Methods for highly multiplexed RNA imaging are limited in spatial resolution and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to the mouse brain, which yielded the readout of thousands of genes, including splice variants. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in the neurons of the mouse hippocampus, revealing patterns across multiple cell types, layer-specific cell types across the mouse visual cortex, and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus, ExSeq enables highly multiplexed mapping of RNAs from nanoscale to system scale.

scholarly journals A Powerful Method for Transcriptional Profiling of Specific Cell Types in Eukaryotes: Laser-Assisted Microdissection and RNA Sequencing

PLoS ONE ◽  
2012 ◽  
Vol 7 (1) ◽  
pp. e29685 ◽  
Author(s):  
Marc W. Schmid ◽  
Anja Schmidt ◽  
Ulrich C. Klostermeier ◽  
Matthias Barann ◽  
Philip Rosenstiel ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Transcriptional Profiling ◽  
Cell Types ◽  
Specific Cell ◽  
Powerful Method

scholarly journals Exo-enzymatic addition of diazirine-modified sialic acid to cell surfaces enables photocrosslinking of glycoproteins

2021 ◽  
Author(s):  
Nageswari Yarravarapu ◽  
Rohit Sai Reddy Konada ◽  
Narek Darabedian ◽  
Nichole J. Pedowtiz ◽  
Soumya N. Krishnamurthy ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Cell Types ◽  
Cell Surfaces ◽  
Binding Interactions ◽  
Multiple Cell ◽  
Labeling Method ◽  
Cell Surface Glycoconjugates

Glycan binding often mediates extracellular macromolecular recognition events. Accurate characterization of these binding interactions can be difficult because of dissociation and scrambling that occur during purification and analysis steps. Use of photocrosslinking methods has been pursued to covalently capture glycan-dependent interactions in situ however use of metabolic glycan engineering methods to incorporate photocrosslinking sugar analogs is limited to certain cell types. Here we report an exo-enzymatic labeling method to add a diazirine-modified sialic acid (SiaDAz) to cell surface glycoconjugates. The method involves chemoenzymatic synthesis of diazirine-modified CMP-sialic acid (CMP-SiaDAz), followed by sialyltransferase-catalyzed addition of SiaDAz to desialylated cell surfaces. Cell surface SiaDAz-ylation is compatible with multiple cell types and is facilitated by endogenous extracellular sialyltransferase activity present in Daudi B cells. This method for extracellular addition of α2-6-linked SiaDAz enables UV-induced crosslinking of CD22, demonstrating the utility for covalent capture of glycan-mediated binding interactions.

scholarly journals Chemogenetic Tools for Causal Cellular and Neuronal Biology

2018 ◽  
Vol 98 (1) ◽  
pp. 391-418 ◽  
Author(s):  
Deniz Atasoy ◽  
Scott M. Sternson
Keyword(s):  
G Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Cell Populations ◽  
Specific Cell ◽  
Cellular Processes ◽  
Distinct Cell ◽  
G Protein Coupled ◽  
Pharmacological Control

Chemogenetic technologies enable selective pharmacological control of specific cell populations. An increasing number of approaches have been developed that modulate different signaling pathways. Selective pharmacological control over G protein-coupled receptor signaling, ion channel conductances, protein association, protein stability, and small molecule targeting allows modulation of cellular processes in distinct cell types. Here, we review these chemogenetic technologies and instances of their applications in complex tissues in vivo and ex vivo.

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