Global Run-On sequencing (GRO-seq) library preparation from Drosophila ovaries

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 timeconsuming, 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 genomewide transcription.