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Real-time analysis of Ttanscription factor binding, transcription, translation, and turnover to display global events during cellular activation.
J. Vis. Exp. 2018:e56752 (2018)
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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Publication type
Article: Journal article
Document type
Scientific Article
Keywords
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
ISSN (print) / ISBN
1940-087X
e-ISSN
1940-087X
Quellenangaben
Volume: 2018,
Issue: 133,
Article Number: e56752
Publisher
JoVE
Publishing Place
1 Alewife Center, Ste 200, Cambridge, Ma 02140 Usa
Non-patent literature
Publications
Reviewing status
Peer reviewed
Institute(s)
Institute of Diabetes and Obesity (IDO)