Design and use of a back splicing junction probe for pulldown of circular RNA-binding proteins in cell lines

Abstract

Summary: Due to the unique structure of circular RNAs, it is challenging to use traditional pulldown approaches. Here, we describe the design and use of a probe that spans the back splicing junction (BSJ), enabling interaction with circular RNAs. The probe repeats four times, allowing efficient and specific pulldown of circular RNAs and their binding partners. This protocol describes the steps for mouse cardiac fibroblast (MCF) cells; we have also verified the protocol in other cell types.For complete details on the use and execution of this protocol, please refer to Wu et al. (2021). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• RNAseq
• Molecular biology
• Molecular/Chemical probes