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Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity
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JoVE Journal Genetik
Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

8,872 Views

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09:21 min

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October 22, 2018

DOI:

10.3791/57982-v

DOI:
10.3791/57982-v

•

09:21 min
October 22, 2018

•

9 Views
,
1Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France, 2Centre National de la Recherche Scientifique, Illkirch, France, 3Institut National de la Santé et de la Recherche Médicale, Illkirch, France, 4Université de Strasbourg

Özet

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The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

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Saccharomyces CerevisiaeMetabolic Labeling4-thiouracilQuantificationNewly Synthesized MRNARNA Polymerase II ActivityMicroarray HybridizationHigh-throughput SequencingS. PombeYPD MediumYAS MediumCell CountingCell CentrifugationRNA ExtractionRNA Polymerase II

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Baptista, T., Devys, D. Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity. J. Vis. Exp. (140), e57982, doi:10.3791/57982 (2018).

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