From high-throughput DNA and RNA sequencing technologies, it is evident that more than two-thirds of the mammalian genome is transcribed and nearly 98% of the transcriptional output in humans constitute noncoding RNA, comprising tens of thousands of small and long noncoding RNAs. These observations have put the study of RNA expression levels at the center of molecular biology research. The transcriptional output of cells changes temporally throughout different cell cycle phases, or in response to a large panel of stimuli. In such instances, the measure of induced RNA transcripts might be obscured by the presence of steady-state RNA levels in the total transcriptome. With this protocol, we provide a method for labeling and purification of the nascent RNAs transcribed over short periods of time in cultured cells. The supplementation of cell culture medium with a chemically modified analog of uridine, ethynyl-uridine, allows for the subsequent biotinylation of ethynyl-uridine residues with a click-chemistry reaction. The labeled RNA is then purified on streptavidin beads and eluted. The purified RNA is suitable for use in RT-qPCR assays as well as in deep sequencing applications.
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This work was supported by the grants from Knut and Alice Wallenberg Foundation [KAW2014.0057]; Swedish Foundation for Strategic Research [RB13-0204]; Swedish Cancer Research foundation [Cancerfonden: Kontrakt no. CAN2018/591]; Swedish Research Council [2017-02834]; Barncancerfonden [PR2018-0090]; Ingabritt Och Arne Lundbergs forskningsstiftelse and LUA/ALF (to C.K.).
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1.Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska AcademyUniversity of GothenburgGothenburgSweden
2.Normandie Univ, UNICAEN, INSERM U1086 “ANTICIPE” (Interdisciplinary Research Unit for Cancers Prevention and Treatment, Axis BioTICLA “Biology and Innovative Therapeutics for Ovarian Cancers”)CaenFrance
3.Comprehensive Cancer Centre François Baclesse, UNICANCERCaenFrance