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

Daniel A LorenzHsuan-Lin HerKylie A ShenKatie RothamelKasey R HuttAllan C NojaderaStephanie C BrunsSergei A ManakovBrian A YeeKaren B ChapmanGene W Yeo
Published in: Nature methods (2022)
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.
Keyphrases
  • nucleic acid
  • binding protein
  • randomized controlled trial
  • circulating tumor
  • gene expression
  • single cell
  • single molecule
  • small molecule
  • high throughput
  • mass spectrometry