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 Chapman, G Yeo
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.