Denaturing cross-linking immunoprecipitation to identify footprints for RNA-binding proteins

Michael Rosenberg; Vered Levy; Verena K Maier; Barry Kesner; Roy Blum; Jeannie T Lee

doi:10.1016/j.xpro.2021.100819

Denaturing cross-linking immunoprecipitation to identify footprints for RNA-binding proteins

STAR Protoc. 2021 Sep 16;2(4):100819. doi: 10.1016/j.xpro.2021.100819. eCollection 2021 Dec 17.

Authors

Michael Rosenberg^{1

2}, Vered Levy^{1

2}, Verena K Maier^{1

2}, Barry Kesner^{1

2}, Roy Blum^{1

2}, Jeannie T Lee^{1

2}

Affiliations

¹ Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA.
² Department of Genetics, Harvard Medical School, Boston, MA 02114, USA.

Abstract

The isolation of protein-RNA complexes in the "denaturing cross-linked RNA immunoprecipitation" (dCLIP) protocol is based on biotin-tagging proteins of interest, UV cross-linking RNA to protein in vivo, RNase protection assay, and isolating RNA-protein complexes under denaturing conditions over a streptavidin column. Insofar as conventional antibody-based CLIP assays have been challenging to apply to Polycomb complexes, dCLIP has been applied successfully and yields small RNA footprints from which de novo motif analysis can be performed to identify RNA binding motifs. For complete details on the use and execution of this protocol, please refer to Rosenberg et al. (2017).

Keywords: Genetics; Genomics; Molecular Biology; Molecular/Chemical Probes; Protein Biochemistry; Systems biology.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
Embryonic Stem Cells
HEK293 Cells
Humans
Immunoprecipitation / methods*
Mice
Protein Footprinting / methods*
RNA* / chemistry
RNA* / genetics
RNA* / metabolism
RNA-Binding Proteins* / chemistry
RNA-Binding Proteins* / genetics
RNA-Binding Proteins* / metabolism
Sequence Analysis, RNA

Substances

RNA-Binding Proteins
RNA

Abstract

Publication types

MeSH terms

Substances

Grants and funding