Protocol for detecting genomic insulators in Drosophila using insulator-seq, a massively parallel reporter assay
Year: 2024
Type: article
Abstract: Genomic insulators are DNA elements that prevent transcriptional activation of a promoter by an enhancer when interposed. We present a protocol for insulator-seq that enables high-throughput screening of genomic insulators using a plasmid-based massively parallel reporter assay in Drosophila cultured cells. We describe steps for insulator reporter plasmid library generation, transient transfection into cultured cells, and sequencing library preparation and provide a pipeline for data analysis. For complete details on the use and execution of this protocol, please refer to Tonelli et al. 1 • Instructions for high-throughput screening of genomic insulators in Drosophila cells • Steps for high-throughput barcoded insulator reporter plasmid library generation • Guidance on DNA- and RNA-seq library preparation and quality control Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Genomic insulators are DNA elements that prevent transcriptional activation of a promoter by an enhancer when interposed. We present a protocol for insulator-seq that enables high-throughput screening of genomic insulators using a plasmid-based massively parallel reporter assay in Drosophila cultured cells. We describe steps for insulator reporter plasmid library generation, transient transfection into cultured cells, and sequencing library preparation and provide a pipeline for data analysis.
Source: STAR Protocols
Authors: Anastasiia Tonelli, Pascal Cousin, Maria Cristina Gambetta
Institution: University of Lausanne
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,500
OpenAlex ID: W4403717234