Murine intestinal cell dissociation suitable for multi-omics single-cell assays v1 (protocols.io.bt9enr3e)

protocols.io ◽  
2021 ◽  
Author(s):  
Mackenzie L ◽  
Astrid Kosters ◽  
Luisa Cervantes-Barragan ◽  
Eliver Ghosn
Keyword(s):  
Single Cell ◽  
Intestinal Cell ◽  
Cell Dissociation ◽  
Cell Assays

scholarly journals Human intestinal cell dissociation suitable for multi-omics single-cell assays v1 (protocols.io.bubrnsm6)

protocols.io ◽  
2021 ◽  
Author(s):  
Astrid Kosters ◽  
Junkai Yang ◽  
Ann Dodd ◽  
Mackenzie L ◽  
Greg not provided ◽  
...  
Keyword(s):  
Single Cell ◽  
Intestinal Cell ◽  
Cell Dissociation ◽  
Cell Assays

scholarly journals Single cell assays unveil functional and transcriptional heterogeneity of human hemopoietic lympho-myeloid progenitors

2017 ◽  
Vol 53 ◽  
pp. S50
Author(s):  
Bilyana Stoilova ◽  
Dimitris Karamitros ◽  
Zahra Aboukhalil ◽  
Andreas Reinisch ◽  
Fiona Hamey ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Assays ◽  
Myeloid Progenitors

scholarly journals Single-cell transcriptomics reveals immune response of intestinal cell types to viral infection

2020 ◽  
Author(s):  
Sergio Triana ◽  
Megan L. Stanifer ◽  
Mohammed Shahraz ◽  
Markus Mukenhirn ◽  
Carmon Kee ◽  
...  
Keyword(s):  
Immune Response ◽  
Single Cell ◽  
Cell Lineage ◽  
Enteric Virus ◽  
Cell Types ◽  
Intestinal Cell ◽  
Intestinal Epithelial ◽  
Proliferating Cells ◽  
Interferon Stimulated Genes ◽  
Human Intestinal Epithelium

AbstractHuman intestinal epithelial cells form a primary barrier protecting us from pathogens, yet only limited knowledge is available about individual contribution of each cell type to mounting an immune response against infection. Here, we developed a pipeline combining single-cell RNA-Seq and highly-multiplex RNA imaging and applied it to human intestinal organoids infected with human astrovirus, a model human enteric virus. We found that interferon controls the infection and that astrovirus infects all major cell types and lineages with a preferential infection of proliferating cells. Intriguingly, each intestinal epithelial cell lineage has a unique basal expression of interferon-stimulated genes and, upon astrovirus infection, undergoes an antiviral transcriptional reprogramming by upregulating distinct sets of interferon-stimulated genes. These findings suggest that in the human intestinal epithelium, each cell lineage plays a unique role in resolving virus infection. Our pipeline can be applicable to other organoids and viruses, opening new avenues to unravel roles of individual cell types in viral pathogenesis.

scholarly journals Convergence of oncogenic cooperation at single-cell and single-gene levels drives leukemic transformation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yuxuan Liu ◽  
Zhimin Gu ◽  
Hui Cao ◽  
Pranita Kaphle ◽  
Junhua Lyu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cancer Progression ◽  
Single Gene ◽  
Myeloid Cell ◽  
Cellular Level ◽  
Integrative Approach ◽  
Cancer Evolution ◽  
Cell Assays

AbstractCancers develop from the accumulation of somatic mutations, yet it remains unclear how oncogenic lesions cooperate to drive cancer progression. Using a mouse model harboring NRasG12D and EZH2 mutations that recapitulates leukemic progression, we employ single-cell transcriptomic profiling to map cellular composition and gene expression alterations in healthy or diseased bone marrows during leukemogenesis. At cellular level, NRasG12D induces myeloid lineage-biased differentiation and EZH2-deficiency impairs myeloid cell maturation, whereas they cooperate to promote myeloid neoplasms with dysregulated transcriptional programs. At gene level, NRasG12D and EZH2-deficiency independently and synergistically deregulate gene expression. We integrate results from histopathology, leukemia repopulation, and leukemia-initiating cell assays to validate transcriptome-based cellular profiles. We use this resource to relate developmental hierarchies to leukemia phenotypes, evaluate oncogenic cooperation at single-cell and single-gene levels, and identify GEM as a regulator of leukemia-initiating cells. Our studies establish an integrative approach to deconvolute cancer evolution at single-cell resolution in vivo.

scholarly journals Simultaneous trimodal single-cell measurement of transcripts, epitopes, and chromatin accessibility using TEA-seq

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Elliott Swanson ◽  
Cara Lord ◽  
Julian Reading ◽  
Alexander T Heubeck ◽  
Palak C Genge ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Single Cell ◽  
Human Peripheral Blood ◽  
Single Cells ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Specific Gene ◽  
Test Case ◽  
Cell Assays ◽  
Paired Measurement

Single-cell measurements of cellular characteristics have been instrumental in understanding the heterogeneous pathways that drive differentiation, cellular responses to signals, and human disease. Recent advances have allowed paired capture of protein abundance and transcriptomic state, but a lack of epigenetic information in these assays has left a missing link to gene regulation. Using the heterogeneous mixture of cells in human peripheral blood as a test case, we developed a novel scATAC-seq workflow that increases signal-to-noise and allows paired measurement of cell surface markers and chromatin accessibility: integrated cellular indexing of chromatin landscape and epitopes, called ICICLE-seq. We extended this approach using a droplet-based multiomics platform to develop a trimodal assay that simultaneously measures transcriptomics (scRNA-seq), epitopes, and chromatin accessibility (scATAC-seq) from thousands of single cells, which we term TEA-seq. Together, these multimodal single-cell assays provide a novel toolkit to identify type-specific gene regulation and expression grounded in phenotypically defined cell types.

Cellular phenotypes of normal and leukemic hemopoietic cells determined by analysis with selected antibody combinations

Blood ◽  
1980 ◽  
Vol 56 (3) ◽  
pp. 430-441 ◽  
Author(s):  
G Janossy ◽  
FJ Bollum ◽  
KF Bradstock ◽  
J Ashley
Keyword(s):  
Bone Marrow ◽  
Monoclonal Antibodies ◽  
Single Cell ◽  
Normal Cell ◽  
Cell Types ◽  
Precursor Cells ◽  
Leukemic Cells ◽  
Double Staining ◽  
Cell Assays ◽  
Cellular Phenotypes

Abstract Individual leukemic cells and the corresponding rare normal cell types in nonleukemic bone marrow were analyzed with various combinations of antisera (labeled with different fluorochromes: TRITC and FITC). Double staining for membrane Ia-like molecules (TRITC) and nuclear terminal transferase (FITC) was a very useful combination that distinguished common non-T, non-B ALL (Ia+,TdT+) and thymic ALL (Ia-,TdT+) from the rare cases of B ALL (Ia+,TdT-) and from AML (frequently Ia+, TdT-; in some cases Ia-, TdT-). Additional antisera (such as anti-ALL, anti- HuTLA, anti-immunoglobulin reagents, etc.) confirmed the diagnosis and further characterized the leukemic blasts. Ia+,TdT+ cells could be observed in low numbers in normal and nonleukemic regenerating marrow and were probably normal precursor cells; this reagent combinations was, therefore, not useful for monitoring residual non-T, non-B ALL blasts in treated patients. Other marker combinations detecting pre-B ALL blasts (double staining for cytoplasmic IgM and nuclear TdT) and Thy-ALL blasts (HuTLA+,TdT+) were, however, virtually leukemia specific in the bone marrow and could be used to effectively monitor residual leukemic cells throughout the disease. These combined single-cell assays are not only economical and informative but are also important for assessing the heterogeneity of leukemia and for standardizing new mouse or rat monoclonal antibodies for diagnosis.

scholarly journals Altered microRNA expression links IL6 and TNF-induced inflammaging with myeloid malignancy in humans and mice

Blood ◽  
2020 ◽  
Vol 135 (25) ◽  
pp. 2235-2251 ◽  
Author(s):  
Jennifer M. Grants ◽  
Joanna Wegrzyn ◽  
Tony Hui ◽  
Kieran O’Neill ◽  
Marion Shadbolt ◽  
...  
Keyword(s):  
Single Cell ◽  
Transcriptome Profiling ◽  
Lymphoid Cells ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Myeloid Malignancy ◽  
Enhanced Sensitivity ◽  
Age Related ◽  
Cell Assays ◽  
Quiescent Hscs

Abstract Aging is associated with significant changes in the hematopoietic system, including increased inflammation, impaired hematopoietic stem cell (HSC) function, and increased incidence of myeloid malignancy. Inflammation of aging (“inflammaging”) has been proposed as a driver of age-related changes in HSC function and myeloid malignancy, but mechanisms linking these phenomena remain poorly defined. We identified loss of miR-146a as driving aging-associated inflammation in AML patients. miR-146a expression declined in old wild-type mice, and loss of miR-146a promoted premature HSC aging and inflammation in young miR-146a–null mice, preceding development of aging-associated myeloid malignancy. Using single-cell assays of HSC quiescence, stemness, differentiation potential, and epigenetic state to probe HSC function and population structure, we found that loss of miR-146a depleted a subpopulation of primitive, quiescent HSCs. DNA methylation and transcriptome profiling implicated NF-κB, IL6, and TNF as potential drivers of HSC dysfunction, activating an inflammatory signaling relay promoting IL6 and TNF secretion from mature miR-146a−/− myeloid and lymphoid cells. Reducing inflammation by targeting Il6 or Tnf was sufficient to restore single-cell measures of miR-146a−/− HSC function and subpopulation structure and reduced the incidence of hematological malignancy in miR-146a−/− mice. miR-146a−/− HSCs exhibited enhanced sensitivity to IL6 stimulation, indicating that loss of miR-146a affects HSC function via both cell-extrinsic inflammatory signals and increased cell-intrinsic sensitivity to inflammation. Thus, loss of miR-146a regulates cell-extrinsic and -intrinsic mechanisms linking HSC inflammaging to the development of myeloid malignancy.

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