Global Run-On Sequencing (GRO-seq) Library Preparation from Drosophila Ovaries.
Methods Mol Biol
; 1328: 217-30, 2015.
Article
em En
| MEDLINE
| ID: mdl-26324441
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Ovário
/
RNA
/
Sequenciamento de Nucleotídeos em Larga Escala
Limite:
Animals
Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article