A5072751630- Single-cell and spatial transcriptomics
- RNA modifications and cancer
- RNA Research and Splicing
- Acute Myeloid Leukemia Research
- Cancer-related molecular mechanisms research
- Cancer Genomics and Diagnostics
- Genomics and Phylogenetic Studies
- Extracellular vesicles in disease
- Molecular Biology Techniques and Applications
- Advanced biosensing and bioanalysis techniques
- Gene expression and cancer classification
- Hematopoietic Stem Cell Transplantation
- Epigenetics and DNA Methylation
- Acute Lymphoblastic Leukemia research
- Immune Cell Function and Interaction
- Genomics and Chromatin Dynamics
- MicroRNA in disease regulation
- RNA and protein synthesis mechanisms
- Immunotherapy and Immune Responses
- Blood disorders and treatments
- Mycobacterium research and diagnosis
- Cellular Mechanics and Interactions
- RNA Interference and Gene Delivery
- Cancer Cells and Metastasis
- Digestive system and related health
Karolinska Institutet
2018-2024
Ludwig-Maximilians-Universität München
2015-2022
Urologische Klinik München
2022
Max Planck Institute for Biology of Ageing
2019
Hospitais da Universidade de Coimbra
2019
University of Coimbra
2019
Genomics (United Kingdom)
2018
Fundación Juan March
2015
Helmholtz Zentrum München
2015
Single-cell RNA-sequencing (scRNA-seq) experiments typically analyze hundreds or thousands of cells after amplification the cDNA. The high throughput is made possible by early introduction sample-specific bar codes (BCs), and bias alleviated unique molecular identifiers (UMIs). Thus, ideal analysis pipeline for scRNA-seq data needs to efficiently tabulate reads according both BC UMI.
The recent rapid spread of single cell RNA sequencing (scRNA-seq) methods has created a large variety experimental and computational pipelines for which best practices have not yet been established. Here, we use simulations based on five scRNA-seq library protocols in combination with nine realistic differential expression (DE) setups to systematically evaluate three mapping, four imputation, seven normalisation testing approaches resulting ~3000 pipelines, allowing us also assess...
Abstract Single-cell RNA sequencing (scRNA-seq) has emerged as a central genome-wide method to characterize cellular identities and processes. Consequently, improving its sensitivity, flexibility, cost-efficiency can advance many research questions. Among the flexible plate-based methods, single-cell barcoding (SCRB-seq) is highly sensitive efficient. Here, we systematically evaluate experimental conditions of this protocol find that adding polyethylene glycol considerably increases...
Abstract Currently, quantitative RNA-seq methods are pushed to work with increasingly small starting amounts of RNA that require amplification. However, it is unclear how much noise or bias amplification introduces and this affects precision accuracy quantification. To assess the effects amplification, reads originated from same molecule (PCR-duplicates) need be identified. Computationally, read duplicates defined by their mapping position, which does not distinguish PCR- natural hence treat...
Abstract Current single-cell RNA sequencing (scRNA-seq) methods with high cellular throughputs sacrifice full-transcript coverage and often sensitivity. Here we describe Smart-seq3xpress, which miniaturizes streamlines the Smart-seq3 protocol to substantially reduce reagent use increase throughput. Smart-seq3xpress analysis of peripheral blood mononuclear cells resulted in a granular atlas complete common rare cell types. Compared droplet-based that sequences ends, additional revealed...
Cell types can be classified according to shared patterns of transcription. Non-genetic variability among individual cells the same type has been ascribed stochastic transcriptional bursting and transient cell states. Using high-coverage single-cell RNA profiling, we asked whether long-term, heritable differences in gene expression impart diversity within type. Studying clonal human lymphocytes mouse brain cells, uncovered a vast different clones vivo. We combined chromatin accessibility...
Significance This study provides a mechanistic explanation for the differentiation of trophoblasts from human pluripotent stem cells, process relying on BMP morphogens. We found that network transcription factors GATA2, GATA3, TFAP2A, and TFAP2C regulates early trophoblast progenitor specification by activating placental genes inhibiting pluripotency gene OCT4 , thus acting to couple with exit pluripotency. To demonstrate relevance our findings in vivo, we show down-regulating GATA3 primate...
Power analysis is essential to optimize the design of RNA-seq experiments and assess compare power detect differentially expressed genes in data. PowsimR a flexible tool simulate evaluate differential expression from bulk especially single-cell data making it suitable for priori posterior analyses.The R package associated tutorial are freely available at https://github.com/bvieth/powsimR.vieth@bio.lmu.de or hellmann@bio.lmu.de.Supplementary Bioinformatics online.
Cost-efficient library generation by early barcoding has been central in propelling single-cell RNA sequencing. Here, we optimize and validate prime-seq, an bulk RNA-seq method. We show that it performs equivalently to TruSeq, a standard method, but is fourfold more cost-efficient due almost 50-fold cheaper costs. also direct isolation step, intronic reads are derived from RNA, compare cost-efficiencies of available protocols. conclude prime-seq currently one the best options set up protocol...
An increasing number of long noncoding RNAs (lncRNAs) have experimentally confirmed functions, yet little is known about their transcriptional dynamics and it challenging to determine regulatory effects. Here, we used allele-sensitive single-cell RNA sequencing demonstrate that, compared messenger RNAs, lncRNAs twice as duration between two bursts. Additionally, observed increased cell-to-cell variability in lncRNA expression due lower frequency bursting producing larger numbers molecules....
Abstract Rare multipotent stem cells replenish millions of blood per second through a time-consuming process, passing multiple stages increasingly lineage-restricted progenitors. Although insults to the blood-forming system highlight need for more rapid replenishment from cells, established models hematopoiesis implicate only one mandatory differentiation pathway each cell lineage. Here, we establish nonhierarchical relationship between distinct that all lineages and almost exclusively...
Formation of tissue-specific transcriptional programs underlies multicellular development, including dorsoventral (DV) patterning the Drosophila embryo. This involves interactions between enhancers and promoters in a chromatin context, but how landscape influences transcription is not fully understood.
Abstract Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. Here, we describe recently evolved pathway in which global hypomethylation achieved by the coupling active passive demethylation. TET activity required, albeit indirectly, for demethylation, mostly occurs at sites devoid binding. Instead, TET-mediated locus-specific necessary activating subset genes, including pluripotency germline marker Dppa3 ( Stella, Pgc7 ). DPPA3 turn...
Abstract Tissues within an organism and even cell types a tissue can age with different velocities. However, it is unclear whether cells of one type experience aging trajectories depending on their spatial location. Here, we used transcriptomics in combination single-cell ATAC-seq RNA-seq, lipidomics functional assays to address how the male murine liver are affected by age-related changes microenvironment. Integration datasets revealed zonation-specific metabolic states, epigenome...
Here, we show that the Wnt5a-haploinsufficient niche regenerates dysfunctional HSCs, which do not successfully engraft in secondary recipients. RNA sequencing of regenerated donor Lin− SCA-1+ KIT+ (LSK) cells shows dysregulated expression ZEB1-associated genes involved small GTPase-dependent actin polymerization pathway. Misexpression DOCK2, WAVE2, and activation CDC42 results apolar F-actin localization, leading to defects adhesion, migration homing HSCs a microenvironment. Moreover, these...
Abstract Single-cell sequencing methods rely on molecule-counting strategies to account for amplification biases, yet no experimental strategy evaluate counting performance exists. Here, we introduce molecular spikes—RNA spike-ins containing built-in unique identifiers (UMIs) that use identify critical and computational conditions accurate RNA in single-cell RNA-sequencing (scRNA-seq). Using spikes, uncovered impaired were not informative cellular abundances due inflated UMI counts. We...