Multiplexed transcriptome discovery of RNA-binding protein binding sites by antibody-barcode eCLIP

Daniel A Lorenz; Hsuan-Lin Her; Kylie A Shen; Katie Rothamel; Kasey R Hutt; Allan C Nojadera; Stephanie C Bruns; Sergei A Manakov; Brian A Yee; Karen B Chapman; Gene W Yeo

doi:10.1038/s41592-022-01708-8

Multiplexed transcriptome discovery of RNA-binding protein binding sites by antibody-barcode eCLIP

Nat Methods. 2023 Jan;20(1):65-69. doi: 10.1038/s41592-022-01708-8. Epub 2022 Dec 22.

Authors

Daniel A Lorenz¹, Hsuan-Lin Her^{2

3

4}, Kylie A Shen¹, Katie Rothamel^{2

3

4}, Kasey R Hutt¹, Allan C Nojadera¹, Stephanie C Bruns¹, Sergei A Manakov¹, Brian A Yee^{2

3

4}, Karen B Chapman⁵, Gene W Yeo^{6

7

8}

Affiliations

¹ Eclipse Bioinnovations, San Diego, CA, USA.
² Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.
³ Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA.
⁴ Stem Cell Program, University of California San Diego, La Jolla, CA, USA.
⁵ Eclipse Bioinnovations, San Diego, CA, USA. karen.chapman@eclipsebio.com.
⁶ Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA. geneyeo@ucsd.edu.
⁷ Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA. geneyeo@ucsd.edu.
⁸ Stem Cell Program, University of California San Diego, La Jolla, CA, USA. geneyeo@ucsd.edu.

Abstract

Ultraviolet crosslinking and immunoprecipitation (CLIP) methodologies enable the identification of RNA binding sites of RNA-binding proteins (RBPs). Despite improvements in the library preparation of RNA fragments, the enhanced CLIP (eCLIP) protocol requires 4 days of hands-on time and lacks the ability to process several RBPs in parallel. We present a new method termed antibody-barcode eCLIP that utilizes DNA-barcoded antibodies and proximity ligation of the DNA oligonucleotides to RBP-protected RNA fragments to interrogate several RBPs simultaneously. We observe performance comparable with that of eCLIP with the advantage of dramatically increased scaling while maintaining the same material requirement of a single eCLIP experiment.

Publication types

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

MeSH terms

Antibodies / chemistry
Binding Sites
Immunoprecipitation
Protein Binding
RNA* / genetics
RNA-Binding Proteins / metabolism
Transcriptome*

Substances

RNA
RNA-Binding Proteins
Antibodies

Grants and funding

T32 CA067754/CA/NCI NIH HHS/United States