scholarly journals Mass Cytometry for Multivariate Organoid Signalling Analysis

2020 ◽  
Author(s):  
Xiao Qin ◽  
Jahangir Sufi ◽  
Petra Vlckova ◽  
Pelagia Kyriakidou ◽  
Sophie E. Acton ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Single Cell Analysis ◽  
Cell Type ◽  
Mass Cytometry ◽  
Post Translational Modification ◽  
Specific Analysis ◽  
Cell Type Specific ◽  
Tissue Models

Abstract Organoids are powerful biomimetic tissue models. Despite their increasing popularity, no existing methods are suitable for cell-type specific analysis of post-translational modification (PTM) signalling networks in organoids. Here we report a multivariate mass cytometry (MC) protocol for single-cell analysis of cell-type specific PTM signalling in organoid monocultures and organoids co-cultured with stromal and immune cells. Thiol-reactive Organoid Barcoding in situ (TOBis) was developed to facilitate high-throughput comparison of signalling networks between organoid cultures. Taken together, our protocol enables high-throughput multivariate PTM signalling analysis of healthy and cancerous organoids at the single-cell level.

scholarly journals Mass Cytometry for Multivariate Organoid Signalling Analysis

2020 ◽  
Author(s):  
Xiao Qin ◽  
Jahangir Sufi ◽  
Petra Vlckova ◽  
Pelagia Kyriakidou ◽  
Sophie E. Acton ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Single Cell Analysis ◽  
Cell Type ◽  
Mass Cytometry ◽  
Post Translational Modification ◽  
Specific Analysis ◽  
Cell Type Specific ◽  
Tissue Models

Abstract Organoids are powerful biomimetic tissue models. Despite their increasing popularity, no existing methods are suitable for cell-type specific analysis of post-translational modification (PTM) signalling networks in organoids. Here we report a multivariate mass cytometry (MC) protocol for single-cell analysis of cell-type specific PTM signalling in organoid monocultures and organoids co-cultured with stromal and immune cells. Thiol-reactive Organoid Barcoding in situ (TOBis) was developed to facilitate high-throughput comparison of signalling networks between organoid cultures. Taken together, our protocol enables high-throughput multivariate PTM signalling analysis of healthy and cancerous organoids at the single-cell level.

scholarly journals Single-Cell Signalling Analysis of Heterocellular Organoids

2019 ◽  
Author(s):  
Xiao Qin ◽  
Jahangir Sufi ◽  
Petra Vlckova ◽  
Pelagia Kyriakidou ◽  
Sophie E. Acton ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Single Cell Analysis ◽  
Cell Signalling ◽  
Tumour Microenvironment ◽  
Cell Type ◽  
Mass Cytometry ◽  
Post Translational Modification ◽  
Stromal Fibroblasts ◽  
Cell Type Specific

Organoids are powerful biomimetic tissue models. Despite their widespread adoption, methods to analyse cell-type specific post-translational modification (PTM) signalling networks in organoids are absent. Here we report multivariate single-cell analysis of cell-type specific signalling networks in organoids and organoid co-cultures. Simultaneous measurement of 28 PTMs in >1 million single small intestinal organoid cells by mass cytometry reveals cell-type and cell-state specific signalling networks in stem, Paneth, enteroendocrine, tuft, goblet cells, and enterocytes. Integrating single-cell PTM analysis with Thiol-reactive Organoid Barcoding in situ (TOBis) enables high-throughput comparison of signalling networks between organoid cultures. Multivariate cell-type specific PTM analysis of colorectal cancer tumour microenvironment organoids reveals that shApc, KrasG12D, and Trp53R172H cell-autonomously mimic signalling states normally induced by stromal fibroblasts and macrophages. These results demonstrate how standard mass cytometry workflows can be modified to perform high-throughput multivariate cell-type specific signalling analysis of healthy and cancerous organoids.

scholarly journals Comprehensive analysis of single cell ATAC-seq data with SnapATAC

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rongxin Fang ◽  
Sebastian Preissl ◽  
Yang Li ◽  
Xiaomeng Hou ◽  
Jacinta Lucero ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Expression Patterns ◽  
Regulatory Elements ◽  
Cellular Heterogeneity ◽  
Specific Gene ◽  
Open Chromatin ◽  
Cell Type ◽  
Process Data ◽  
Cell Type Specific

AbstractIdentification of the cis-regulatory elements controlling cell-type specific gene expression patterns is essential for understanding the origin of cellular diversity. Conventional assays to map regulatory elements via open chromatin analysis of primary tissues is hindered by sample heterogeneity. Single cell analysis of accessible chromatin (scATAC-seq) can overcome this limitation. However, the high-level noise of each single cell profile and the large volume of data pose unique computational challenges. Here, we introduce SnapATAC, a software package for analyzing scATAC-seq datasets. SnapATAC dissects cellular heterogeneity in an unbiased manner and map the trajectories of cellular states. Using the Nyström method, SnapATAC can process data from up to a million cells. Furthermore, SnapATAC incorporates existing tools into a comprehensive package for analyzing single cell ATAC-seq dataset. As demonstration of its utility, SnapATAC is applied to 55,592 single-nucleus ATAC-seq profiles from the mouse secondary motor cortex. The analysis reveals ~370,000 candidate regulatory elements in 31 distinct cell populations in this brain region and inferred candidate cell-type specific transcriptional regulators.

scholarly journals Spatiotemporal mapping of RNA editing in the developing mouse brain using in situ sequencing reveals regional and cell-type-specific regulation

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Elin Lundin ◽  
Chenglin Wu ◽  
Albin Widmark ◽  
Mikaela Behm ◽  
Jens Hjerling-Leffler ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Editing ◽  
Mouse Brain ◽  
Developmental Stages ◽  
Cell Types ◽  
Specific Cell ◽  
Cell Type ◽  
Cell Type Specific ◽  
Specific Regulation

Abstract Background Adenosine-to-inosine (A-to-I) RNA editing is a process that contributes to the diversification of proteins that has been shown to be essential for neurotransmission and other neuronal functions. However, the spatiotemporal and diversification properties of RNA editing in the brain are largely unknown. Here, we applied in situ sequencing to distinguish between edited and unedited transcripts in distinct regions of the mouse brain at four developmental stages, and investigate the diversity of the RNA landscape. Results We analyzed RNA editing at codon-altering sites using in situ sequencing at single-cell resolution, in combination with the detection of individual ADAR enzymes and specific cell type marker transcripts. This approach revealed cell-type-specific regulation of RNA editing of a set of transcripts, and developmental and regional variation in editing levels for many of the targeted sites. We found increasing editing diversity throughout development, which arises through regional- and cell type-specific regulation of ADAR enzymes and target transcripts. Conclusions Our single-cell in situ sequencing method has proved useful to study the complex landscape of RNA editing and our results indicate that this complexity arises due to distinct mechanisms of regulating individual RNA editing sites, acting both regionally and in specific cell types.

scholarly journals Single-cell Stereo-seq enables cell type-specific spatial transcriptome characterization in Arabidopsis leaves

2021 ◽  
Author(s):  
Keke Xia ◽  
Hai-Xi Sun ◽  
Jie Li ◽  
Jiming Li ◽  
Yu Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Spatial Information ◽  
Transcriptome Profiling ◽  
Cell Type ◽  
Spatially Resolved ◽  
Cell Type Specific

Understanding the complex functions of plant leaves requires spatially resolved gene expression profiling with single-cell resolution. However, although in situ gene expression profiling technologies have been developed, this goal has not yet been achieved. Here, we present the first in situ single-cell transcriptome profiling in plant, scStereo-seq (single-cell SpaTial Enhanced REsolution Omics-sequencing), which enabled the bona fide single-cell spatial transcriptome of Arabidopsis leaves. We successfully characterized subtle but significant transcriptomic differences between upper and lower epidermal cells. Furthermore, with high-resolution location information, we discovered the cell type-specific spatial gene expression gradients from main vein to leaf edge. By reconstructing those spatial gradients, we show for the first time the distinct spatial developmental trajectories of vascular cells and guard cells. Our findings show the importance of incorporating spatial information for answering complex biological questions in plant, and scStereo-seq offers a powerful single cell spatially resolved transcriptomic strategy for plant biology.

scholarly journals Single-cell analysis of Foxp1-driven mechanisms essential for striatal development

2019 ◽  
Author(s):  
Ashley G. Anderson ◽  
Ashwinikumar Kulkarni ◽  
Matthew Harper ◽  
Genevieve Konopka
Keyword(s):  
Single Cell ◽  
Target Genes ◽  
Single Cell Analysis ◽  
Cell Types ◽  
Brain Regions ◽  
Projection Neurons ◽  
Loss Of Function ◽  
Cell Type ◽  
Motor Information ◽  
Cell Type Specific

AbstractThe striatum is a critical forebrain structure for integrating cognitive, sensory, and motor information from diverse brain regions into meaningful behavioral output. However, the transcriptional mechanisms that underlie striatal development and organization at single-cell resolution remain unknown. Here, we show that Foxp1, a transcription factor strongly linked to autism and intellectual disability, regulates organizational features of striatal circuitry in a cell-type-dependent fashion. Using single-cell RNA-sequencing, we examine the cellular diversity of the early postnatal striatum and find that cell-type-specific deletion ofFoxp1in striatal projection neurons alters the cellular composition and neurochemical architecture of the striatum. Importantly, using this approach, we identify the non-cell autonomous effects produced by disruptingFoxp1in one cell-type and the molecular compensation that occurs in other populations. Finally, we identify Foxp1-regulated target genes within distinct cell-types and connect these molecular changes to functional and behavioral deficits relevant to phenotypes described in patients withFOXP1loss-of-function mutations. These data reveal cell-type-specific transcriptional mechanisms underlying distinct features of striatal circuitry and identify Foxp1 as a key regulator of striatal development.

scholarly journals Lanthanide nanoparticles for high sensitivity multiparameter single cell analysis

2019 ◽  
Vol 10 (10) ◽  
pp. 2965-2974 ◽  
Author(s):  
Jothirmayanantham Pichaandi ◽  
Guangyao Zhao ◽  
Alexandre Bouzekri ◽  
Elsa Lu ◽  
Olga Ornatsky ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Single Cell Analysis ◽  
High Sensitivity ◽  
Cell Analysis ◽  
Mass Cytometry ◽  
Analytical Technique ◽  
In Cells

Mass cytometry (MC) is a high throughput multiparameter analytical technique for determining biomarker expression in cells.

In situ High-Throughput Single-Cell Analysis Reveals the Crosstalk between Nanoparticle-Induced Cell Responses

2021 ◽  
Vol 55 (8) ◽  
pp. 5136-5142
Author(s):  
Yuanyuan Wang ◽  
Fengbang Wang ◽  
Zihan Chen ◽  
Maoyong Song ◽  
Xinglei Yao ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Single Cell Analysis ◽  
Cell Analysis ◽  
Cell Responses

86 Co-detection of RNA and protein in FFPE tumor samples by combining RNAscope in situ hybridization and immunohistochemistry assays

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A95-A95
Author(s):  
Anushka Dikshit ◽  
Xiao-jun Ma ◽  
Emerald Doolittle ◽  
Lydia Hernandez ◽  
Jyoti Sheldon ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
In Situ Hybridization ◽  
Single Cell ◽  
Immune Cells ◽  
Cell Type ◽  
Ffpe Tumor ◽  
Cell Type Specific ◽  
Co Detection

BackgroundSpatially resolved gene expression has emerged as a crucial technique to understand complex multicellular interactions within the tumor and its microenvironment. Interrogation of complex cellular interactions within the tumor microenvironment (TME) requires a multi-omics approach where multiple RNA and protein targets can be visualized within the same tumor sample and be feasible in FFPE sample types. Simultaneous detection of RNA and protein can reveal cellular sources of secreted proteins, identify specific cell types, and visualize the spatial organization of cells within the tissue. Examination of RNA by in situ hybridization (ISH) and protein by immunohistochemistry (IHC) or immunofluorescence (IF) are widely used and accepted techniques for the detection of biomarkers in tumor samples. Given the similarities in workflow, co-detection of RNA and protein by combining ISH and IHC/IF in a single assay can be a powerful multi-omics solution for interrogating the complex tumor and its microenvironment.MethodsIn this report we combined the single cell, single molecule RNA ISH technology known as RNAscope with IHC/IF to simultaneously detect RNA and protein in the same FFPE tumor section using both chromogenic and fluorescence detection methods.ResultsWe demonstrate co-localization of target mRNA and the corresponding protein in human cancer samples, visualize infiltration of immune cells into the TME, characterize the activation state of immune cells in the TME, identify single cell gene expression within cellular boundaries demarcated by IHC/IF, examine cell type-specific expression of multiple immune checkpoint markers, and distinguish endogenous T cells from activated CAR+ T cells. Overall, we show that co-detection of RNA by the RNAscope ISH assay and protein by the IHC/IF assay in the same FFPE section is a feasible methodology. The combined RNAscope ISH-IHC/IF workflow is a powerful technique that can be used to study gene expression signatures at the RNA and protein level with spatial and single cell resolution.ConclusionsBy leveraging the strength of the similar workflows of RNAscope ISH and IHC/IF assays, this methodology combines transcriptomics and proteomics in the same tissue section, providing a multi-omics approach for characterizing complex tissues and revealing cell type specific gene expression with spatial and single cell resolution.

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