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

Feiya Li; Nan Wu; Qiwei Yang; William W Du; Javeria Qadir; Burton B Yang

doi:10.1016/j.xpro.2022.101702

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

STAR Protoc. 2022 Dec 16;3(4):101702. doi: 10.1016/j.xpro.2022.101702. Epub 2022 Sep 22.

Authors

Feiya Li¹, Nan Wu², Qiwei Yang², William W Du², Javeria Qadir², Burton B Yang³

Affiliations

¹ Sunnybrook Research Institute, and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada. Electronic address: feiya.li@mail.utoronto.ca.
² Sunnybrook Research Institute, and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
³ Sunnybrook Research Institute, and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada. Electronic address: byang@sri.utoronto.ca.

Abstract

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).

Keywords: Molecular biology; Molecular/Chemical probes; RNAseq.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Line
Mice
RNA Splicing
RNA* / metabolism
RNA, Circular*
RNA-Binding Proteins / metabolism

Substances

RNA, Circular
RNA
RNA-Binding Proteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding