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

• Published in: Nature methods Vol. 20; no. 1; pp. 65 - 69
• Main Authors: Lorenz, Daniel A, Her, Hsuan-Lin, Shen, Kylie A, Rothamel, Katie, Hutt, Kasey R, Nojadera, Allan C, Bruns, Stephanie C, Manakov, Sergei A, Yee, Brian A, Chapman, Karen B, Yeo, Gene W
• Format: Journal Article
• Language: English
• Published: United States Nature Publishing Group 01.01.2023; Nature Publishing Group US
• Subjects: Antibodies; Antibodies - chemistry; Bar codes; Binding Sites; Brief Communication; Crosslinking; Deoxyribonucleic acid; DNA; Fragments; Gene expression; Immunoprecipitation; Medical research; Multiplexing; Oligonucleotides; Protein Binding; Proteins; Ribonucleic acid; RNA; RNA - genetics; RNA-binding protein; RNA-Binding Proteins - metabolism; Transcriptome; Transcriptomes
• ISSN: 1548-7091; 1548-7105
• DOI: 10.1038/s41592-022-01708-8

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.