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A Multiplexed Barcodelet Single-Cell RNA-Seq Approach Elucidates Combinatorial Signaling Pathways that Drive ESC Differentiation.

ABSTRACT: Empirical optimization of stem cell differentiation protocols is time consuming, is laborintensive, and typically does not comprehensively interrogate all relevant signaling pathways. Here we describe barcodelet single-cell RNA sequencing (barRNA-seq), which enables systematic exploration of cellular perturbations by tagging individual cells with RNA "barcodelets" to identify them on the basis of the treatments they receive. We apply barRNA-seq to simultaneously manipulate up to seven developmental pathways and study effects on embryonic stem cell (ESC) germ layer specification and mesodermal specification, uncovering combinatorial effects of signaling pathway activation on gene expression. We further develop a data-driven framework for identifying combinatorial signaling perturbations that drive cells toward specific fates, including several annotated in an existing scRNA-seq gastrulation atlas, and use this approach to guide ESC differentiation into a notochord-like population. We expect that barRNA-seq will have broad utility for investigating and understanding how cooperative signaling pathways drive cell fate acquisition.

SUBMITTER: Yeo GHT

PROVIDER: S-EPMC7398619 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A Multiplexed Barcodelet Single-Cell RNA-Seq Approach Elucidates Combinatorial Signaling Pathways that Drive ESC Differentiation

Project description:Empirical optimization of stem cell differentiation protocols is time consuming, is labor intensive, and typi- cally does not comprehensively interrogate all relevant signaling pathways. Here we describe barcodelet sin- gle-cell RNA sequencing (barRNA-seq), which enables systematic exploration of cellular perturbations by tagging individual cells with RNA ‘‘barcodelets’’ to identify them on the basis of the treatments they receive. We apply barRNA-seq to simultaneously manipulate up to seven developmental pathways and study effects on embryonic stem cell (ESC) germ layer specification and mesodermal specification, uncovering combina- torial effects of signaling pathway activation on gene expression. We further develop a data-driven frame- work for identifying combinatorial signaling perturbations that drive cells toward specific fates, including several annotated in an existing scRNA-seq gastrulation atlas, and use this approach to guide ESC differen- tiation into a notochord-like population. We expect that barRNA-seq will have broad utility for investigating Q2 and understanding how cooperative signaling pathways drive cell fate acquisition.

2020-05-26 | GSE122009 | GEO

A Multiplexed Barcodelet Single-Cell RNA-Seq Approach Elucidates Combinatorial Signaling Pathways that Drive ESC Differentiation

Project description:A Multiplexed Barcodelet Single-Cell RNA-Seq Approach Elucidates Combinatorial Signaling Pathways that Drive ESC Differentiation

| PRJNA503119 | ENA

CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq.

Project description:Single-cell transcriptomics requires a method that is sensitive, accurate, and reproducible. Here, we present CEL-Seq2, a modified version of our CEL-Seq method, with threefold higher sensitivity, lower costs, and less hands-on time. We implemented CEL-Seq2 on Fluidigm's C1 system, providing its first single-cell, on-chip barcoding method, and we detected gene expression changes accompanying the progression through the cell cycle in mouse fibroblast cells. We also compare with Smart-Seq to demonstrate CEL-Seq2's increased sensitivity relative to other available methods. Collectively, the improvements make CEL-Seq2 uniquely suited to single-cell RNA-Seq analysis in terms of economics, resolution, and ease of use.

| S-EPMC4848782 | biostudies-literature

Decoding Neuronal Diversification by Multiplexed Single-cell RNA-Seq.

Project description:Cellular reprogramming is driven by a defined set of transcription factors; however, the regulatory logic that underlies cell-type specification and diversification remains elusive. Single-cell RNA-seq provides unprecedented coverage to measure dynamic molecular changes at the single-cell resolution. Here, we multiplex and ectopically express 20 pro-neuronal transcription factors in human dermal fibroblasts and demonstrate a widespread diversification of neurons based on cell morphology and canonical neuronal marker expressions. Single-cell RNA-seq analysis reveals diverse and distinct neuronal subtypes, including reprogramming processes that strongly correlate with the developing brain. Gene mapping of 20 exogenous pro-neuronal transcription factors further unveiled key determinants responsible for neuronal lineage specification and a regulatory logic dictating neuronal diversification, including glutamatergic and cholinergic neurons. The multiplex scRNA-seq approach is a robust and scalable approach to elucidate lineage and cellular specification across various biological systems.

| S-EPMC8072034 | biostudies-literature

Identification of Molecular Biomarkers and Key Pathways for Esophageal Carcinoma (EsC): A Bioinformatics Approach.

Project description:Esophageal carcinoma (EsC) is a member of the cancer group that occurs in the esophagus; globally, it is known as one of the fatal malignancies. In this study, we used gene expression analysis to identify molecular biomarkers to propose therapeutic targets for the development of novel drugs. We consider EsC associated four different microarray datasets from the gene expression omnibus database. Statistical analysis is performed using R language and identified a total of 1083 differentially expressed genes (DEGs) in which 380 are overexpressed and 703 are underexpressed. The functional study is performed with the identified DEGs to screen significant Gene Ontology (GO) terms and associated pathways using the Database for Annotation, Visualization, and Integrated Discovery repository (DAVID). The analysis revealed that the overexpressed DEGs are principally connected with the protein export, axon guidance pathway, and the downexpressed DEGs are principally connected with the L13a-mediated translational silencing of ceruloplasmin expression, formation of a pool of free 40S subunits pathway. The STRING database used to collect protein-protein interaction (PPI) network information and visualize it with the Cytoscape software. We found 10 hub genes from the PPI network considering three methods in which the interleukin 6 (IL6) gene is the top in all methods. From the PPI, we found that identified clusters are associated with the complex I biogenesis, ubiquitination and proteasome degradation, signaling by interleukins, and Notch-HLH transcription pathway. The identified biomarkers and pathways may play an important role in the future for developing drugs for the EsC.

| S-EPMC8769846 | biostudies-literature

ePathOptimize: A Combinatorial Approach for Transcriptional Balancing of Metabolic Pathways.

Project description:The ability to fine tune gene expression has created the field of metabolic pathway optimization and balancing where a variety of factors affecting flux balance are carefully modulated to improve product titers, yields, and productivity. Using a library of isopropyl ?-D-1-thiogalactopyranoside (IPTG)-inducible mutant T7 promoters of varied strength a combinatorial method was developed for transcriptional balancing of the violacein pathway. Violacein biosynthesis involves a complex five-gene pathway that is an excellent model for exploratory metabolic engineering efforts into pathway regulation and control due to many colorful intermediates and side products allowing for easy analysis and strain comparison. Upon screening approximately 4% of the total initial library, several high-titer mutants were discovered that resulted in up to a 63-fold improvement over the control strain. With further fermentation optimization, titers were improved to 1829 ± 46 mg/L; a 2.6-fold improvement in titer and a 30-fold improvement in productivity from previous literature reports.

| S-EPMC4650668 | biostudies-literature

MiRNAs Expression Profiling in Raw264.7 Macrophages after Nfatc1-Knockdown Elucidates Potential Pathways Involved in Osteoclasts Differentiation.

Project description:Differentiation of macrophages toward osteoclasts is crucial for bone homeostasis but can be detrimental in disease states, including osteoporosis and cancer. Therefore, understanding the osteoclast differentiation process and the underlying regulatory mechanisms may facilitate the identification of new therapeutic targets. Hereby, we tried to reveal new miRNAs potentially involved in the regulation of early steps of osteoclastogenesis, with a particular focus on those possibly correlated with NFATc1 expression, by studying miRNAs profiling. During the first 24 h of osteoclastogenesis, 38 miRNAs were differentially expressed between undifferentiated and RANKL-stimulated RAW264.7 cells, while 10 miRNAs were differentially expressed between RANKL-stimulated cells transfected with negative control or NFATc1-siRNAs. Among others, the expression levels of miR-411, miR-144 and members of miR-29, miR-30, and miR-23 families changed after RANKL stimulation. Moreover, the potential role of miR-124 during osteoclastogenesis was explored by transient cell transfection with anti-miR-124 or miR-124-mimic. Two relatively unknown miRNAs, miR-880-3p and miR-295-3p, were differentially expressed between RANKL-stimulated/wild-type and RANKL-stimulated/NFATc1-silenced cells, suggesting their possible correlation with NFATc1. KEGG enrichment analyses showed that kinase and phosphatase enzymes were among the predicted targets for many of the studied miRNAs. In conclusion, our study provides new data on the potential role and possible targets of new miRNAs during osteoclastogenesis.

| S-EPMC8614811 | biostudies-literature

Self-assembled combinatorial encoding nanoarrays for multiplexed biosensing.

Project description:Multiplexed and sensitive detection of nucleic acids, proteins, or other molecules from a single solution and a small amount of sample is of great demand in biomarker profiling and disease diagnostics. Here we describe a new concept using combinatorial self-assembly of DNA nanotiles into micrometer-sized two-dimensional arrays that carry nucleic acid probes and barcoded fluorescent dyes to achieve multiplexed detection. We demonstrated the specificity and sensitivity of the arrays by detecting multiple DNA sequences and aptamer binding molecules. This DNA tile-array-based sensor platform can be constructed through DNA self-assembly. The attachment of different molecular probes can be achieved by simple DNA hybridization so bioconjugation is not necessary for the labeling. Accurate control of the interprobe distances and solution-based binding reactions ensures fast target binding kinetics.

| S-EPMC1963466 | biostudies-literature

Single cell RNA-Seq reveals pre-cDCs fate determined by transcription factor combinatorial dose.

Project description:BackgroundClassic dendritic cells (cDCs) play a central role in the immune system by processing and presenting antigens to activate T cells, and consist of two major subsets: CD141+ cDC (cDC1) and CD1c+ cDC (cDC2). A population of migratory precursor cells, the pre-cDCs, is the immediate precursors to both cDC subsets. Previous studies showed that there were two pre-committed pre-cDC subpopulations. However, the key molecular drivers of pre-commitment in human pre-cDCs were not investigated.ResultsTo identify the key molecular drivers for pre-commitment in human pre-cDCs, we performed single cell RNA sequencing (RNA-Seq) of two cDC subsets and pre-cDCs, and bulk RNA-Seq of pre-cDCs and cDCs from human peripheral blood. We found that pre-DC subpopulations cannot be separated by either variable genes within pre-cDCs or differentially expressed genes between cDC1 and cDC2. In contrast, they were separated by 16 transcription factors that are themselves differentially expressed or have regulated targets enriched in the differentially expressed genes between bulk cDC1 and cDC2, with one subpopulation close to cDC1 and the other close to cDC2. More importantly, these two pre-cDC sub-populations are correlated with ratio of IRF8 to IRF4 expression level more than their individual expression level. We also verified these findings using three recently published datasets.ConclusionsIn this study, we demonstrate that single cell transcriptome profiling can reveal pre-cDCs differentiation map, and our results suggest the concept that combinatorial dose of transcription factors determines cell differentiation fate.

| S-EPMC6599345 | biostudies-literature

Efficient combinatorial targeting of RNA transcripts in single cells with Cas13 RNA Perturb-seq.

Project description:Pooled CRISPR screens coupled with single-cell RNA-sequencing have enabled systematic interrogation of gene function and regulatory networks. Here, we introduce Cas13 RNA Perturb-seq (CaRPool-seq), which leverages the RNA-targeting CRISPR-Cas13d system and enables efficient combinatorial perturbations alongside multimodal single-cell profiling. CaRPool-seq encodes multiple perturbations on a cleavable CRISPR array that is associated with a detectable barcode sequence, allowing for the simultaneous targeting of multiple genes. We compared CaRPool-seq to existing Cas9-based methods, highlighting its unique strength to efficiently profile combinatorially perturbed cells. Finally, we apply CaRPool-seq to perform multiplexed combinatorial perturbations of myeloid differentiation regulators in an acute myeloid leukemia (AML) model system and identify extensive interactions between different chromatin regulators that can enhance or suppress AML differentiation phenotypes.

| S-EPMC10030154 | biostudies-literature

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