Abstract
In the past decade, deep-sequencing approaches have greatly improved our knowledge of the genome’s potential and have become a crucial milestone for new discoveries in genomics. Transcription is the first step of gene expression; therefore, the detection and measurement of transcription rates is of great interest. Here, a detailed protocol for global run-on sequencing (GRO-seq) library preparation from Drosophila ovaries is described. The method relies on rapid isolation of nuclei with halted transcription, then restarting transcription in physiological conditions in the presence of a labeled nucleotide. The newly transcribed nascent RNA is then isolated and cloned using a small RNA cloning protocol. Although it is time-consuming, the global run-on method allows the user to profile the position, orientation and amount of transcriptionally engaged RNA polymerases across the genome, therefore providing a snapshot of genome-wide transcription.
Key words
- Drosophila
- Germline
- GRO-seq
- Nuclear Run-On
- Nascent RNA
- Transcriptome
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Acknowledgments
I thank J. Lis and A. Kalmykova laboratories for their efforts on improving GRO-seq and run-on protocols, respectively, which influenced the present work. I am grateful to Leah Sabin for critical reading of the manuscript, language editing, and helpful suggestions.
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Rozhkov, N.V. (2015). Global Run-On Sequencing (GRO-seq) Library Preparation from Drosophila Ovaries. In: Bratu, D., McNeil, G. (eds) Drosophila Oogenesis. Methods in Molecular Biology, vol 1328. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2851-4_16
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DOI: https://doi.org/10.1007/978-1-4939-2851-4_16
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2850-7
Online ISBN: 978-1-4939-2851-4
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