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.
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA, nik.rozhkov@gmail.com.
Nikolay V
NV
Rozhkov
D000818:Animals; D004331:Drosophila melanogaster; D005260:Female; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D010053:Ovary / Q000378:metabolism*; D012313:RNA / Q000235:genetics*
D012313:RNA