Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

An accessible and convenient assay to profile single-cell newly synthesized transcriptome reveals transcription factors and regulon dynamics during early-stage T-cell activation

ABSTRACT: Sequencing newly synthesized transcriptome in addition to the regular transcriptome in single cells enable the study of gene expression temporal dynamics during rapid chromatin and gene regulation processes. However, current single-cell newly synthesized transcriptome assays require in-house technology expertise to achieve a high cellular throughput, preventing their widespread application. Here, we develop NOTE-seq to simultaneously profile regular and newly synthesized transcriptome in single cells. NOTE-seq combines 4-thiouridine labeling of newly synthesized RNA, thiol-alkylation-based chemical conversion, and the streamlined 10X Genomics platform, offering a high cellular throughput accessible to and convenient for regular biology labs without single-cell technology expertise. Using NOTE-seq, we investigate the temporal dynamics of gene expression during early-stage T-cell activation in human Jurkat T cells and mouse naïve T cells, characterize transcription factors and regulons, and discover Fli-1 as a master transcription factor for gene regulation upon T-cell activation. Interestingly, Fli-1 level in T cells is sensitive to the treatment of camptothecin, a topoisomerase inhibitor used in cancer chemotherapy, indicating its potential complication on the immune system.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE270292 | GEO | 2024/08/23

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

NASC-seq monitors RNA synthesis in single cells

Project description:Sequencing of newly synthesized RNA can monitor transcriptional dynamics with great sensitivity and high temporal resolution, but is currently restricted to populations of cells. Here, we develop new transcriptome alkylation-dependent single-cell RNA sequencing (NASC-seq), to monitor newly synthesized and pre-existing RNA in single cells. We validate the method on pre-alkylated RNA, and by demonstrating that more newly synthesized RNA was detected for genes with known high mRNA turnover. NASC-seq reveals rapidly up- and down-regulated genes during T-cell activation, and RNA arising from induced genes is essentially only newly synthesized. The newly synthesized and pre-existing transcriptomes after T-cell activation are distinct, confirming that we simultaneously measure gene expression at two time points in single cells. Altogether, NASC-seq is an accessible and powerful tool to investigate transcriptional dynamics that enables the precise monitoring of RNA synthesis at flexible time periods during homeostasis, perturbation responses and cellular differentiation.

2019-07-18 | GSE128273 | GEO

Characterizing the temporal dynamics of gene expression in single cells with sci-fate

Project description:Gene expression is a dynamic process on multiple scales, e.g. the cell cycle, response to stimuli, normal differentiation and development, etc. However, nearly all techniques for profiling gene expression in single cells fail to directly capture these temporal dynamics, which limits the scope of biology that can be effectively investigated. Towards addressing this, we developed sci-fate, a new technique that combines S4U labeling of newly synthesized mRNA with single cell combinatorial indexing (sci-), in order to concurrently profile the whole and newly synthesized transcriptome in each of many single cells. As a proof-of-concept, we applied sci-fate to a model system of cortisol response, and characterized expression dynamics in over 6,000 single cells. From these data, we quantify the dynamics of the cell cycle and of glucocorticoid receptor activation, while also exploring their intersection. We furthermore use these data to develop a framework for estimating cell state transition probabilities, and to identify factors whose dynamic expression potentially regulates these transitions. The experimental and computational methods described here may be broadly applicable to quantitatively characterize cell state dynamics in in vitro systems.

2020-04-12 | GSE131351 | GEO

Mycobacterial strain-specific temporal modulations of newly synthesized macrophage secretome

Project description:THP-1 macrophages were infected with four strains of Mycobacterium tuberculosis to study the temporal dynamics of newly synthesized proteins in the secretome. Temporal snapshots of secretome reflect the macrophage response to pathogenicity which in combination with intracellular events, completes the disease picture. However, such studies are compromised by limitations of quantitative proteomics. Metabolic labeling by SILAC allows a 3-plex experiment while isobaric chemical labeling by iTRAQ/TMT allows up to 8 to 10-plex respectively. This makes studying temporal proteome dynamics an intangible and elusive proposition. We have developed a new variant of hyperplexing method, combining triplex SILAC with 6-plex iTRAQ to achieve 18-plex quantitation in a single MS run. THP-1 macrophages were infected with H37Ra, H37Rv, BND433 and JAL2287 and the newly synthesized secreted host proteins were studied over six temporal frame still 30 hours post infection, at a difference of 4 hours each. For quantitation, the strains were encoded with two sets of triple SILAC- H37Ra & H37Rv in one and BND433 & JAL2287 in another with a control in each. These sets were then iTRAQ labeled to encode for temporal profiles across six time points in 6-plex iTRAQ. Effectively a 36-plex design with 4 replicates of each set, these experiments were completed within few days on the mass spectrometer. Using MaxQuant and in house developed tools and pipelines, we have analysed the data to map the temporal and strain specific dynamics of newly synthesized proteins in host. Hyperplexing enables large scale spatio-temporal systems biology studies where large number of samples can be processed simultaneously and in quantitative manner.

2016-08-10 | PXD004281 | Pride

a6A-seq: N6-allyladenosine-based cellular messenger RNA metabolic labelling and sequencing

Project description:In this work, we applied a6A to label and sequence cellular mRNA in an immunoprecipitation-free and mutation-based manner, termed a6A-seq. a6A-seq was utilized to study the change of cellular gene expression profile under a methionine-free stressed condition. Compared with regular RNA-seq, a6A-seq could more sensitively detect the level change of newly synthesized mRNAs.

2021-12-31 | GSE192818 | GEO

Time- and Polarity-dependent Proteomic Changes associated with Homeostatic Scaling at Central Synapses

Project description:In homeostatic scaling, the cellular mechanisms that detect the offset from the set-point, the duration of the offset and implement a cellular response are not well-understood. To understand the time-dependent dynamics,we manipulated activity for 2 hrs to induce the process of up or down-scaling and metabolically labelled nascent proteins using BONCAT. We analyzed the newly synthesized proteins that exhibited significant increases or decreases in expression in response to activity manipulations and identified 168 proteins. Then, to obtain a temporal trajectory of the cellular response, we compared the proteins synthesized within 2 and 24 hrs of an activity manipulation. Surprisingly, there was little overlap in the significantly regulated newly synthesized proteins identified in the early- and late-response datasets. There was, however, overlap in the functional categories that are modulated early and late, indicating that within protein function groups, different proteomic choices can be made to effect early and late homeostatic responses.

2018-02-16 | PXD008271 | Pride

Temporal dynamics of macrophage heterogeneity after myocardial infarction

Project description:In this study, we attempted to identify the heterogeneity and temporal dynamics of leukocytes at single-cell level in mouse heart after inducing MI using the longitudinal single-cell RNA sequencing and spatial transcriptome sequencing.

2022-06-19 | GSE163129 | GEO

SaptioTemporal dynamics of macrophage heterogeneity after myocardial infarction

2022-06-19 | GSE165857 | GEO

Native Prohibitin Complex Enrichment

Project description:Folding of newly synthesized proteins poses challenges for a functional proteome. Dedicated protein quality control (PQC) systems promote either the folding of nascent polypeptides at ribosomes or, if this fails, ensure their degradation. While well studied for cytosolic protein biogenesis, it is not understood how these processes work for mitochondrially-encoded proteins, key subunits of the oxidative phosphorylation system (OXPHOS). Here, we identify dedicated hubs in proximity to mitoribosomal tunnel exits coordinating mitochondrial translation, protein import, assembly, and protein turnover. Conserved prohibitin/m-AAA protease supercomplexes and the availability of assembly chaperones determine the fate of newly synthesized proteins by molecular triaging. The localization of these competing activities in vicinity to the mitoribosomal tunnel exit allows for a prompt decision whether newly synthesized proteins are fed into OXPHOS assembly or degraded. This repository contains the results of the FLAG-based enrichment of the native prohibitin complex. Please note that we measured elution either with LDS sample buffer or using FLAG-peptides. In the published study, the LDS elution buffer was shown.

2023-08-14 | PXD043671 | Pride

Well-TEMP-seq characterizes the temporal dynamics of single-cell gene expression

Project description:Single-cell RNA sequencing reveals the transcriptional heterogeneity of single cells, but offers static snapshots of gene expression and fails to reveal the temporal dynamics of transcription. Herein, we develop Well-TEMP-seq, a high-throughput, cost-effective, accurate, and efficient method for massively parallel profiling the time-resolved dynamics of single-cell gene expression. Well-TEMP-seq combines metabolic RNA labeling with our recently developed microwell-based single-cell RNA sequencing method (Well-Paired-seq) to distinguish newly transcribed RNAs marked by T-to-C substitutions from pre-existing RNAs in each of thousands of single cells. We further apply Well-TEMP-seq to profiling the dynamics of gene expression of colorectal cancer cells exposed to low-dose of 5-AZA-CdR, a clinically used DNA-demethylating anti-tumor drug. Well-TEMP-seq also for the first time reveals the activation of interferon responsive genes by 5-AZA-CdR induced viral mimicry in the first three days after treatment. Well-TEMP-seq will be broadly applicable to unveil the dynamics of single-cell gene expression in diverse biological processes.

2022-02-01 | GSE194357 | GEO

Nascent glycoproteome reveal the suppressed expression of glycosylated LAMP2 in response to N-linked glycosylation inhibition

Project description:(1) Analysis of newly synthesized proteins under glycosylation inhibition (2) Analysis of newly synthesized glycoproteins during NGI-1 treatment

2021-09-04 | PXD028212 | iProX

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

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