Browse
Submit Data
Databases
API
Help

Dataset Information

27 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

STRT-seq-2i: dual-index 5' single cell and nucleus RNA-seq on an addressable microwell array.

ABSTRACT: Single-cell RNA-seq has become routine for discovering cell types and revealing cellular diversity, but archived human brain samples still pose a challenge to current high-throughput platforms. We present STRT-seq-2i, an addressable 9600-microwell array platform, combining sampling by limiting dilution or FACS, with imaging and high throughput at competitive cost. We applied the platform to fresh single mouse cortical cells and to frozen post-mortem human cortical nuclei, matching the performance of a previous lower-throughput platform while retaining a high degree of flexibility, potentially also for other high-throughput applications.

SUBMITTER: Hochgerner H

PROVIDER: S-EPMC5703850 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

STRT-seq-2i: dual-index 5' single cell and nucleus RNA-seq on an addressable microwell array.

Hochgerner Hannah H Lönnerberg Peter P Hodge Rebecca R Mikes Jaromir J Heskol Abeer A Hubschle Hermann H Lin Philip P Picelli Simone S La Manno Gioele G Ratz Michael M Dunne Jude J Husain Syed S Lein Ed E Srinivasan Maithreyan M Zeisel Amit A Linnarsson Sten S

Scientific reports 20171127 1

Single-cell RNA-seq has become routine for discovering cell types and revealing cellular diversity, but archived human brain samples still pose a challenge to current high-throughput platforms. We present STRT-seq-2i, an addressable 9600-microwell array platform, combining sampling by limiting dilution or FACS, with imaging and high throughput at competitive cost. We applied the platform to fresh single mouse cortical cells and to frozen post-mortem human cortical nuclei, matching the performanc ...[more]

PMID: 29180631

Similar Datasets

An Automated Microwell Platform for Large-Scale Single Cell RNA-Seq.

Project description:Recent developments have enabled rapid, inexpensive RNA sequencing of thousands of individual cells from a single specimen, raising the possibility of unbiased and comprehensive expression profiling from complex tissues. Microwell arrays are a particularly attractive microfluidic platform for single cell analysis due to their scalability, cell capture efficiency, and compatibility with imaging. We report an automated microwell array platform for single cell RNA-Seq with significantly improved performance over previous implementations. We demonstrate cell capture efficiencies of >50%, compatibility with commercially available barcoded mRNA capture beads, and parallel expression profiling from thousands of individual cells. We evaluate the level of cross-contamination in our platform by both tracking fluorescent cell lysate in sealed microwells and with a human-mouse mixed species RNA-Seq experiment. Finally, we apply our system to comprehensively assess heterogeneity in gene expression of patient-derived glioma neurospheres and uncover subpopulations similar to those observed in human glioma tissue.

| S-EPMC5037380 | biostudies-literature

Massively parallel single-nucleus RNA-seq with DroNc-seq.

Project description:Single-nucleus RNA sequencing (sNuc-seq) profiles RNA from tissues that are preserved or cannot be dissociated, but it does not provide high throughput. Here, we develop DroNc-seq: massively parallel sNuc-seq with droplet technology. We profile 39,111 nuclei from mouse and human archived brain samples to demonstrate sensitive, efficient, and unbiased classification of cell types, paving the way for systematic charting of cell atlases.

| S-EPMC5623139 | biostudies-literature

A novel addressable 9600-microwell array single cell RNA-seq method applied on fresh mouse cortical cells and frozen human cortical nuclei

Project description:We adopted the STRT-seq [Islam et al., Nat Methods 11, 163-166 (2013)] RNA-seq technology to a 9600-well array and applied it to analyze single cells from mouse and human cortex single cells.

2017-12-06 | GSE101601 | GEO

Single-nucleus RNA-seq identifies Huntington disease astrocyte states.

Project description:Huntington Disease (HD) is an inherited movement disorder caused by expanded CAG repeats in the Huntingtin gene. We have used single nucleus RNASeq (snRNASeq) to uncover cellular phenotypes that change in the disease, investigating single cell gene expression in cingulate cortex of patients with HD and comparing the gene expression to that of patients with no neurological disease. In this study, we focused on astrocytes, although we found significant gene expression differences in neurons, oligodendrocytes, and microglia as well. In particular, the gene expression profiles of astrocytes in HD showed multiple signatures, varying in phenotype from cells that had markedly upregulated metallothionein and heat shock genes, but had not completely lost the expression of genes associated with normal protoplasmic astrocytes, to astrocytes that had substantially upregulated glial fibrillary acidic protein (GFAP) and had lost expression of many normal protoplasmic astrocyte genes as well as metallothionein genes. When compared to astrocytes in control samples, astrocyte signatures in HD also showed downregulated expression of a number of genes, including several associated with protoplasmic astrocyte function and lipid synthesis. Thus, HD astrocytes appeared in variable transcriptional phenotypes, and could be divided into several different "states", defined by patterns of gene expression. Ultimately, this study begins to fill the knowledge gap of single cell gene expression in HD and provide a more detailed understanding of the variation in changes in gene expression during astrocyte "reactions" to the disease.

| S-EPMC7029580 | biostudies-literature

Spatial resolution of single-cell exocytosis by microwell-based individually addressable thin film ultramicroelectrode arrays.

Project description:We report the fabrication and characterization of microwell-based individually addressable microelectrode arrays (MEAs) and their application to spatially and temporally resolved detection of neurotransmitter release across a single pheochromocytoma (PC12) cell. The microwell-based MEAs consist of 16 4-?m-width square ultramicroelectrodes, 25 3-?m-width square ultramicroelectrodes, or 36 2-?m-width square ultramicroelectrodes, all inside a 40 × 40 ?m square SU-8 microwell. MEAs were fabricated on glass substrates by photolithography, thin film deposition, and reactive ion etching. The ultramicroelectrodes in each MEA are tightly defined in a 30 × 30 ?m square area, which is further encased inside the SU-8 microwell. With this method, we demonstrate that these microelectrodes are stable, reproducible, and demonstrate good electrochemical properties using cyclic voltammetry. Effective targeting and culture of a single cell is achieved by combining cell-sized microwell trapping and cell-picking micropipet techniques. The surface of the microelectrodes in the MEA was coated with collagen IV to promote cell adhesion and further single-cell culture, as good adhesion between the cell membrane and the electrode surface is critical for the quality of the measurements. Imaging the spatial distribution of exocytosis at the surface of a single PC12 cell has also been demonstrated with this system. Exocytotic signals have been successfully recorded from eight independent 2-?m-wide ultramicroelectrodes from a single PC12 cell showing that the subcellular heterogeneity in single-cell exocytosis can be precisely analyzed with these microwell-based MEAs.

| S-EPMC4014142 | biostudies-literature

Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons.

Project description:Single-cell RNA sequencing (RNA-Seq) provides rich information about cell types and states. However, it is difficult to capture rare dynamic processes, such as adult neurogenesis, because isolation of rare neurons from adult tissue is challenging and markers for each phase are limited. Here, we develop Div-Seq, which combines scalable single-nucleus RNA-Seq (sNuc-Seq) with pulse labeling of proliferating cells by 5-ethynyl-2'-deoxyuridine (EdU) to profile individual dividing cells. sNuc-Seq and Div-Seq can sensitively identify closely related hippocampal cell types and track transcriptional dynamics of newborn neurons within the adult hippocampal neurogenic niche, respectively. We also apply Div-Seq to identify and profile rare newborn neurons in the adult spinal cord, a noncanonical neurogenic region. sNuc-Seq and Div-Seq open the way for unbiased analysis of diverse complex tissues.

| S-EPMC5480621 | biostudies-literature

Single-nucleus RNA-seq dissection of choroid plexus tumor cell heterogeneity

Project description:The genomic, genetic and cellular events regulating the onset, growth and survival of rare, choroid plexus neoplasms remain poorly understood. Here, we examine the heterogeneity of human choroid plexus tumors by single-nucleus transcriptome analysis of 23,906 cells from four disease-free choroid plexus and eleven choroid plexus tumors. The resulting expression atlas profiles cellular and transcriptional diversity, copy number alterations, and cell-cell interaction networks in normal and cancerous choroid plexus. In choroid plexus tumor epithelial cells, we observe transcriptional changes that correlate with genome-wide methylation profiles. We further characterize tumor type-specific stromal microenvironments that include altered macrophage and mesenchymal cell states, as well as changes in extracellular matrix components. This first single-cell dataset resource from such scarce samples should be valuable for divising therapies against these little-studied neoplasms.

| S-SCDT-10_1038-S44318-024-00283-2 | biostudies-other

Trapping cells on a stretchable microwell array for single-cell analysis.

Project description:There is a need for a technology that can be incorporated into routine laboratory procedures to obtain a continuous, quantitative, fluorescence-based measurement of the dynamic behaviors of numerous individual living cells in parallel, while allowing other manipulations, such as staining, rinsing, and even retrieval of targeted cells. Here, we report a simple, low-cost microarray platform that can trap cells for dynamic single-cell analysis of mammalian cells. The elasticity of polydimethylsiloxane (PDMS) was utilized to trap tens of thousands of cells on an array. The PDMS microwell array was stretched by a tube through which cells were loaded on the array. Cells were trapped on the array by removal of the tube and relaxation of the PDMS. Once that was accomplished, the cells remained trapped on the array without continuous application of an external force and permitted subsequent manipulations, such as staining, rinsing, imaging, and even isolation of targeted cells. We demonstrate the utility of this platform by multicolor analysis of trapped cells and monitoring in individual cells real-time calcium flux after exposure to the calcium ionophore ionomycin. Additionally, a proof of concept for target cell isolation was demonstrated by using a microneedle to locally deform the PDMS membrane in order to retrieve a particular cell from the array.

| S-EPMC3249509 | biostudies-literature

Single-nucleus RNA-seq identifies divergent populations of FSHD2 myotube nuclei.

Project description:FSHD is characterized by the misexpression of DUX4 in skeletal muscle. Although DUX4 upregulation is thought to be the pathogenic cause of FSHD, DUX4 is lowly expressed in patient samples, and analysis of the consequences of DUX4 expression has largely relied on artificial overexpression. To better understand the native expression profile of DUX4 and its targets, we performed bulk RNA-seq on a 6-day differentiation time-course in primary FSHD2 patient myoblasts. We identify a set of 54 genes upregulated in FSHD2 cells, termed FSHD-induced genes. Using single-cell and single-nucleus RNA-seq on myoblasts and differentiated myotubes, respectively, we captured, for the first time, DUX4 expressed at the single-nucleus level in a native state. We identified two populations of FSHD myotube nuclei based on low or high enrichment of DUX4 and FSHD-induced genes ("FSHD-Lo" and "FSHD Hi", respectively). FSHD-Hi myotube nuclei coexpress multiple DUX4 target genes including DUXA, LEUTX and ZSCAN4, and also upregulate cell cycle-related genes with significant enrichment of E2F target genes and p53 signaling activation. We found more FSHD-Hi nuclei than DUX4-positive nuclei, and confirmed with in situ RNA/protein detection that DUX4 transcribed in only one or two nuclei is sufficient for DUX4 protein to activate target genes across multiple nuclei within the same myotube. DUXA (the DUX4 paralog) is more widely expressed than DUX4, and depletion of DUXA suppressed the expression of LEUTX and ZSCAN4 in late, but not early, differentiation. The results suggest that the DUXA can take over the role of DUX4 to maintain target gene expression. These results provide a possible explanation as to why it is easier to detect DUX4 target genes than DUX4 itself in patient cells and raise the possibility of a self-sustaining network of gene dysregulation triggered by the limited DUX4 expression.

| S-EPMC7224571 | biostudies-literature

Single-nucleus RNA-seq identifies transcriptional heterogeneity in multinucleated skeletal myofibers.

Project description:While the majority of cells contain a single nucleus, cell types such as trophoblasts, osteoclasts, and skeletal myofibers require multinucleation. One advantage of multinucleation can be the assignment of distinct functions to different nuclei, but comprehensive interrogation of transcriptional heterogeneity within multinucleated tissues has been challenging due to the presence of a shared cytoplasm. Here, we utilized single-nucleus RNA-sequencing (snRNA-seq) to determine the extent of transcriptional diversity within multinucleated skeletal myofibers. Nuclei from mouse skeletal muscle were profiled across the lifespan, which revealed the presence of distinct myonuclear populations emerging in postnatal development as well as aging muscle. Our datasets also provided a platform for discovery of genes associated with rare specialized regions of the muscle cell, including markers of the myotendinous junction and functionally validated factors expressed at the neuromuscular junction. These findings reveal that myonuclei within syncytial muscle fibers possess distinct transcriptional profiles that regulate muscle biology.

| S-EPMC7733460 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data