Global Run-On Sequencing (GRO-seq) Library Preparation from Drosophila Ovaries
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 wordsDrosophila Germline GRO-seq Nuclear Run-On Nascent RNA Transcriptome
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.
- 11.Churchman LS, Weissman JS (2012) Native elongating transcript sequencing (NET-seq). Curr Protoc Mol Biol Chapter 4;Unit 4, 14, 11–17Google Scholar