scholarly journals scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells

2017 ◽  
Author(s):  
Stephen J. Clark ◽  
Ricard Argelaguet ◽  
Chantriolnt-Andreas Kapourani ◽  
Thomas M. Stubbs ◽  
Heather J. Lee ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Cell ◽  
Single Cells ◽  
Embryonic Stem ◽  
Transcriptome Profiling ◽  
Chromatin Accessibility ◽  
Open Chromatin ◽  
Dynamic Coupling ◽  
Single Cell Sequencing ◽  
Molecular Layers

AbstractParallel single-cell sequencing protocols represent powerful methods for investigating regulatory relationships, including epigenome-transcriptome interactions. Here, we report a novel single-cell method for parallel chromatin accessibility, DNA methylation and transcriptome profiling. scNMT-seq (single-cell nucleosome, methylation and transcription sequencing) uses a GpC methyltransferase to label open chromatin followed by bisulfite and RNA sequencing. We validate scNMT-seq by applying it to differentiating mouse embryonic stem cells, finding links between all three molecular layers and revealing dynamic coupling between epigenomic layers during differentiation.

scholarly journals X Chromosome Dosage Modulates Multiple Molecular and Cellular Properties of Mouse Pluripotent Stem Cells Independently of Global DNA Methylation Levels

2018 ◽  
Author(s):  
Juan Song ◽  
Adrian Janiszewski ◽  
Natalie De Geest ◽  
Lotte Vanheer ◽  
Irene Talon ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
X Chromosome ◽  
Pluripotent Stem Cells ◽  
Gene Dosage ◽  
Embryonic Stem ◽  
Chromatin Accessibility ◽  
Global Dna Methylation ◽  
Open Chromatin ◽  
Mammalian Development

ABSTRACTDuring early mammalian development, the two X-chromosomes in female cells are active. Dosage compensation between XX female and XY male cells is then achieved by X-chromosome inactivation in female cells. Reprogramming female mouse somatic cells into induced pluripotent stem cells (iPSCs) leads to X-chromosome reactivation. The extent to which increased X-chromosome dosage (X-dosage) in female iPSCs leads to differences in the molecular and cellular properties of XX and XY iPSCs is still unclear. We show that chromatin accessibility in mouse iPSCs is modulated by X-dosage. Specific sets of transcriptional regulator motifs are enriched in chromatin with increased accessibility in XX or XY iPSCs. We show that the transcriptome, growth and pluripotency exit are also modulated by X-dosage in iPSCs. To understand the mechanisms by which increased X-dosage modulates the molecular and cellular properties of mouse pluripotent stem cells, we used heterozygous deletions of the X-linked gene Dusp9 in XX embryonic stem cells. We show that X-dosage regulates the transcriptome, open chromatin landscape, growth and pluripotency exit largely independently of global DNA methylation. Our results uncover new insights into X-dosage in pluripotent stem cells, providing principles of how gene dosage modulates the epigenetic and genetic mechanisms regulating cell identity.

P16.09 Regulation of chromatin accessibility in the hypoxic tumor microenvironment of glioblastoma

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii57-ii57
Author(s):  
M Dzwigonska ◽  
J Mieczkowski ◽  
P Pilanc ◽  
S Cyranowski ◽  
A Kominek ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Single Cell ◽  
Cancer Cells ◽  
Single Cells ◽  
Therapy Resistance ◽  
Chromatin Accessibility ◽  
Open Chromatin ◽  
Dependent Manner ◽  
Protein Markers ◽  
Hypoxic Tumor

Abstract BACKGROUND Chromatin structure is often dysregulated in cancers, including glioblastoma (GBM), the most aggressive type of primary brain tumor. GBM has the poorest prognosis with no efficient cure to date due to diffusive growth into the brain, resistance to treatments and the immunosuppressive tumor microenvironment (TME). The growth and invasiveness of GBM is supported by the heterogeneous TME including local microglia and bone-marrow-derived macrophages (collectively known as glioma-associated microglia and macrophages, GAMs). In addition, tumor hypoxia is a key factor in the progression of GBM, as it can globally and rapidly alter gene expression, induce cancer cell invasiveness, stemness and lead to therapy resistance. Hypoxia can influence the pro-tumorigenic function of GAMs by inducing the expression of cytokines and cell surface receptors. However, little is known on the hypoxia-imposed chromatin changes of GAMs and GBM cells, which can in turn impact the interaction between these cell populations. Here we analyze these changes using a single-cell method, which preserves in situ hypoxia within the TME of GBM. MATERIAL AND METHODS Single-cell Pi-ATAC-seq (Protein-indexed Assay of Transposase Accessible Chromatin with sequencing) method in a GL261 murine glioma model was used to simultaneously assess genome-wide chromatin accessibility and expression of intracellular protein markers in single cells, enabling accurate selection of hypoxic and non-hypoxic tumor cells and GAMs. Pi-ATAC-seq is used on paraformaldehyde-perfused tumors and therefore allows capturing unaltered hypoxia-dependent cellular states, that often become distorted during dissociation and preparation of fresh material in most common single-cell methods. RESULTS We optimized Pi-ATAC method in a GL261 GBM mouse model, with specific sorting of GAMs using CD11b+ immunosorting followed by separation of microglia and macrophages, based on intensity of CD45 staining. HIF-1α induction and binding of pimonidazole were used to mark hypoxic populations. Currently, we are investigating the chromatin accessibility profiles of cancer cells and GAMs within the hypoxic tumor microenvironment of GBM. Exploring open chromatin profiles in GAMs and glioma-microglia co-cultures will allow to unravel the mechanisms of chromatin accessibility modulation in the oxygen-dependent manner. CONCLUSION In summary, we optimized the Pi-ATAC method in a mouse GBM model to characterize the chromatin openness changes in GAMs and cancer cells in response to hypoxic stress. Further validation of these results will provide the potential to identify novel markers for GAMs/glioma interactions in hypoxic GBMs and develop novel therapeutic targets.

scholarly journals UniPath: a uniform approach for pathway and gene-set based analysis of heterogeneity in single-cell epigenome and transcriptome profiles

2020 ◽  
Author(s):  
Smriti Chawla ◽  
Sudhagar Samydurai ◽  
Say Li Kong ◽  
Zhenxun Wang ◽  
Wai Leong TAM ◽  
...  
Keyword(s):  
Single Cell ◽  
Expression Profiles ◽  
Single Cells ◽  
Gene Expression Profiles ◽  
Transcriptome Profiling ◽  
Mouse Cell ◽  
Drop Out ◽  
Open Chromatin ◽  
Gene Set Enrichment ◽  
Gene Set

Abstract Recent advances in single-cell open-chromatin and transcriptome profiling have created a challenge of exploring novel applications with a meaningful transformation of read-counts, which often have high variability in noise and drop-out among cells. Here, we introduce UniPath, for representing single-cells using pathway and gene-set enrichment scores by a transformation of their open-chromatin or gene-expression profiles. The robust statistical approach of UniPath provides high accuracy, consistency and scalability in estimating gene-set enrichment scores for every cell. Its framework provides an easy solution for handling variability in drop-out rate, which can sometimes create artefact due to systematic patterns. UniPath provides an alternative approach of dimension reduction of single-cell open-chromatin profiles. UniPath's approach of predicting temporal-order of single-cells using their pathway enrichment scores enables suppression of covariates to achieve correct order of cells. Analysis of mouse cell atlas using our approach yielded surprising, albeit biologically-meaningful co-clustering of cell-types from distant organs. By enabling an unconventional method of exploiting pathway co-occurrence to compare two groups of cells, our approach also proves to be useful in inferring context-specific regulations in cancer cells. Available at https://reggenlab.github.io/UniPathWeb/.

scholarly journals Linking transcriptome and chromatin accessibility in nanoliter droplets for single-cell sequencing

2019 ◽  
Author(s):  
Song Chen ◽  
Blue B Lake ◽  
Kun Zhang
Keyword(s):  
Mrna Expression ◽  
Single Cell ◽  
Adult Mouse ◽  
Single Cells ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Single Cell Sequencing ◽  
Single Nucleus

Linked profiling of transcriptome and chromatin accessibility from single cells can provide unprecedented insights into cellular status. Here we developed a droplet-based Single-Nucleus chromatin Accessibility and mRNA Expression sequencing (SNARE-seq) assay, that we used to profile neonatal and adult mouse cerebral cortices. To demonstrate the strength of single-cell dual-omics profiling, we reconstructed transcriptome and epigenetic landscapes of cell types, uncovered lineage-specific accessible sites, and connected dynamics of promoter accessibility with transcription during neurogenesis.

scholarly journals Manifold alignment reveals correspondence between single cell transcriptome and epigenome dynamics

2017 ◽  
Author(s):  
Joshua D. Welch ◽  
Alexander J. Hartemink ◽  
Jan F. Prins
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Epigenetic Modification ◽  
Single Cells ◽  
Embryonic Stem ◽  
Genomic Data ◽  
Chromatin Accessibility ◽  
Data Sets ◽  
Cell Transcriptome ◽  
Dynamic Interplay

AbstractSingle cell genomic techniques promise to yield key insights into the dynamic interplay between gene expression and epigenetic modification. However, the experimental difficulty of performing multiple measurements on the same cell currently limits efforts to combine multiple genomic data sets into a united picture of single cell variation. We show that it is possible to construct cell trajectories, reflecting the changes that occur in a sequential biological process, from single cell ATAC-seq, bisulfite sequencing, and ChIP-seq data. In addition, we present an approach called MATCHER that computationally circumvents the experimental difficulties inherent in performing multiple genomic measurements on a single cell by inferring correspondence between single cell transcriptomic and epigenetic measurements performed on different cells of the same type. MATCHER works by first learning a separate manifold for the trajectory of each kind of genomic data, then aligning the manifolds to infer a shared trajectory in which cells measured using different techniques are directly comparable. Using scM&T-seq data, we confirm that MATCHER accurately predicts true single cell correlations between DNA methylation and gene expression without using known cell correspondence information. We also used MATCHER to infer correlations among gene expression, chromatin accessibility, and histone modifications in single mouse embryonic stem cells. These results reveal the dynamic interplay between epigenetic changes and gene expression underlying the transition from pluripotency to differentiation priming. Our work is a first step toward a united picture of heterogeneous transcriptomic and epigenetic states in single cells.

scholarly journals Simultaneous multiplexed amplicon sequencing and transcriptome profiling in single cells

2018 ◽  
Author(s):  
Mridusmita Saikia ◽  
Philip Burnham ◽  
Sara H. Keshavjee ◽  
Michael F. Z. Wang ◽  
Michael Heyang ◽  
...  
Keyword(s):  
Infected Cell ◽  
Single Cell ◽  
B Lymphocytes ◽  
Light Chain ◽  
Single Cells ◽  
Variable Region ◽  
Transcriptome Profiling ◽  
Amplicon Sequencing ◽  
Single Cell Sequencing ◽  
Rna Targeting

AbstractWe describe Droplet Assisted RNA Targeting by single cell sequencing (DART-seq), a versatile technology that enables multiplexed amplicon sequencing and transcriptome profiling in single cells. We applied DART-seq to simultaneously characterize the non-A-tailed transcripts of a segmented dsRNA virus and the transcriptome of the infected cell. In addition, we used DART-seq to simultaneously determine the natively paired, variable region heavy and light chain amplicons and the transcriptome of B lymphocytes.

scholarly journals Single-cell sequencing techniques from individual to multiomics analyses

2020 ◽  
Vol 52 (9) ◽  
pp. 1419-1427
Author(s):  
Yukie Kashima ◽  
Yoshitaka Sakamoto ◽  
Keiya Kaneko ◽  
Masahide Seki ◽  
Yutaka Suzuki ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Experimental Methods ◽  
Chromatin Accessibility ◽  
Data Sets ◽  
Transcriptome Data ◽  
Detailed Understanding ◽  
Single Cell Sequencing ◽  
Single Cell Rna Sequencing ◽  
Molecular Profiles

Abstract Here, we review single-cell sequencing techniques for individual and multiomics profiling in single cells. We mainly describe single-cell genomic, epigenomic, and transcriptomic methods, and examples of their applications. For the integration of multilayered data sets, such as the transcriptome data derived from single-cell RNA sequencing and chromatin accessibility data derived from single-cell ATAC-seq, there are several computational integration methods. We also describe single-cell experimental methods for the simultaneous measurement of two or more omics layers. We can achieve a detailed understanding of the basic molecular profiles and those associated with disease in each cell by utilizing a large number of single-cell sequencing techniques and the accumulated data sets.

scholarly journals Single cell transcriptome profiling of mouse and hESC-derived pancreatic progenitors

2018 ◽  
Author(s):  
Nicole A. J. Krentz ◽  
Michelle Lee ◽  
Eric E. Xu ◽  
Shugo Sasaki ◽  
Francis C. Lynn
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Differential Expression Analysis ◽  
Embryonic Stem ◽  
Transcriptome Profiling ◽  
Β Cells ◽  
Pancreatic Progenitors ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome ◽  
Endocrine Progenitors

SummaryHuman embryonic stem cells (hESCs) are a potential unlimited source of insulin-producing β-cells for diabetes treatment. A greater understanding of how β-cells form during embryonic development will improve current hESC differentiation protocols. As β-cells are formed from NEUROG3-expressing endocrine progenitors, this study focused on characterizing the single-cell transcriptomes of mouse and hESC-derived endocrine progenitors. To do this, 7,223 E15.5 and 6,852 E18.5 single cells were isolated from Neurog3-Cre; Rosa26mT/mG embryos, allowing for enrichment of endocrine progenitors (yellow; tdTomato + EGFP) and endocrine cells (green; EGFP). From a NEUROG3-2A-eGFP CyT49 hESC reporter line (N5-5), 4,497 hESC-derived endocrine progenitor cells were sequenced. Differential expression analysis reveals enrichment of markers that are consistent with progenitor, endocrine, or novel cell-state populations. This study characterizes the single-cell transcriptomes of mouse and hESC-derived endocrine progenitors and serves as a resource (https://lynnlab.shinyapps.io/embryonic_pancreas/) for improving the formation of functional β-like cells from hESCs.

scholarly journals Unveiling transposable element expression heterogeneity in cell fate regulation at the single-cell level

2020 ◽  
Author(s):  
Jiangping He ◽  
Isaac A. Babarinde ◽  
Li Sun ◽  
Shuyang Xu ◽  
Ruhai Chen ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Single Cells ◽  
Embryonic Stem ◽  
Cell Type ◽  
Cell Level ◽  
Single Cell Sequencing ◽  
Processing Pipeline ◽  
Sequencing Technologies ◽  
Disease Specific

AbstractTransposable elements (TEs) make up a majority of a typical eukaryote’s genome, and contribute to cell heterogeneity and fate in unclear ways. Single cell-sequencing technologies are powerful tools to explore cells, however analysis is typically gene-centric and TE activity has not been addressed. Here, we developed a single-cell TE processing pipeline, scTE, and report the activity of TEs in single cells in a range of biological contexts. Specific TE types were expressed in subpopulations of embryonic stem cells and were dynamically regulated during pluripotency reprogramming, differentiation, and embryogenesis. Unexpectedly, TEs were expressed in somatic cells, including human disease-specific TEs that are undetectable in bulk analyses. Finally, we applied scTE to single cell ATAC-seq data, and demonstrate that scTE can discriminate cell type using chromatin accessibly of TEs alone. Overall, our results reveal the dynamic patterns of TEs in single cells and their contributions to cell fate and heterogeneity.

Sign in / Sign up

Export Citation Format

Share Document