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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
Bölümler
- 00:04Başlık
- 00:41Cell Culturing
- 01:374tU Labeling with S. pombe as a Spike-in
- 03:20RNA Extraction and DNase Treatment
- 04:30Thiol-specific Biotinylation of Newly Synthesized RNA
- 06:05Purification of Newly Synthesized Fraction from Total and Unlabeled RNA Using Streptavidin-coated Magnetic Beads
- 07:18Results: Quantification of Newly Synthesized mRNA as a Proxy for RNA Polymerase II Activity
- 08:52Conclusion
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.
