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

Lorenz, D. A.; Shen, K. A.; Her, H.-L.; Rothamel, K.; Hutt, K. R.; Nojadera, A. C.; Bruns, S. C.; Manakov, S. A.; Chapman, K. B.; Yeo, G. W.

doi:10.1101/2022.06.08.495357

bioRxiv · 10.1101/2022.06.08.495357

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

Lorenz, D. A.·Shen, K. A.·Her, H.-L.·Rothamel, K.·Hutt, K. R.·Nojadera, A. C.·Bruns, S. C.·Manakov, S. A.·Chapman, K. B.·Yeo, G. W.

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Abstract

UV cross-linking 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 current enhanced CLIP (eCLIP) protocol requires 4 days of hands-on time and lacks the ability to process many RBPs in parallel. We present a new method termed antibody-barcode eCLIP (ABC) that utilizes DNA-barcoded antibodies and proximity ligation of the DNA oligonucleotides to RBP-protected RNA fragments to interrogate multiple RBPs simultaneously. We observe performance comparable to eCLIP with the advantage of a reduced hands-on time of 2 days and dramatically increased scaling while minimizing sample-to-sample variation and maintaining the same material requirement of a single eCLIP experiment.

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Lorenz, D. A., Shen, K. A., Her, H.-L., Rothamel, K., Hutt, K. R., Nojadera, A. C., Bruns, S. C., Manakov, S. A., Chapman, K. B., Yeo, G. W.. 2022-06-09. Multiplexed transcriptome discovery of RNA binding protein binding sites by antibody-barcode eCLIP. https://doi.org/10.1101/2022.06.08.495357

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