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Davari, K. ; Lichti, J. ; Friedel, C.C.* ; Glasmacher, E.

Real-time analysis of Ttanscription factor binding, transcription, translation, and turnover to display global events during cellular activation.

J. Vis. Exp. 2018:e56752 (2018)
Verlagsversion DOI PMC
Open Access Hybrid
Creative Commons Lizenzvertrag
Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up-or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.
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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Schlagwörter Genetics ; Issue 133 ; 4-thiouridine ; Ribosome Profiling ; Chip-seq ; Rna-seq ; Newly Transcribed Rna ; Genome-wide ; Rna Processing ; Splicing ; Cotranscriptional Splicing ; Translation Rate ; Turnover Rate ; Transcription Factor Binding; Nucleotide Resolution; Rna; Reveals; Protein; Seq; Efficient; Genes; Cells
Sprache englisch
Veröffentlichungsjahr 2018
HGF-Berichtsjahr 2018
ISSN (print) / ISBN 1940-087X
e-ISSN 1940-087X
Zeitschrift Journal of Visualized Experiments
Quellenangaben Band: 2018, Heft: 133, Seiten: , Artikelnummer: e56752 Supplement: ,
Verlag JoVE
Verlagsort 1 Alewife Center, Ste 200, Cambridge, Ma 02140 Usa
Begutachtungsstatus Peer reviewed
Institut(e) Institute of Diabetes and Obesity (IDO)
POF Topic(s) 90000 - German Center for Diabetes Research
Forschungsfeld(er) Helmholtz Diabetes Center
PSP-Element(e) G-501900-226
DOI 10.3791/56752
WOS ID WOS:000443329500077
Scopus ID 85044754653
PubMed ID 29578509
Erfassungsdatum 2018-06-05
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