irCLIP

irCLIP

irCLIP maps protein-RNA interaction sites using less sample material, time, and increased cDNA library quality compared to previous CLIP methods. irCLIP was designed to tackle the issues in both iCLIP and HITS-CLIP, such as reverse-transcriptase halting and short cDNA library fragments, by using on-bead nuclease digestion.

First, RNA-protein complexes are UV-crosslinked and immunoprecipitated. Next, they are digested using RNase A and RNase I to excise both ends of the protein-bound RNA strand. An IR800-biotin adapter is ligated to the 3' end of the RNA fragment. After size selection, nitrocellulose blotting, and proteinase K digestion, the RNA strands of interest are purified. They are reverse-transcribed and immunopurified once again. The cDNA strands are circularized and PCR-amplified to produce cDNA libraries for sequencing.

Pros:

• Nonisotopic detection of protein-RNA interactions with minimal starting material
• On-bead nuclease digestion improves the length of RNA fragments for cDNA library prep
• Able to accommodate small cell samples and reveal novel RBP binding sites
• Uses thermostable reverse transcriptase at 60°C, reducing biases by melting secondary RNA structures¹

Cons:

• Not widely adapted by the scientific community yet
• Circularization step may introduce artifacts

• irCLIP: Zarnegar B. J., Flynn R. A., Shen Y., Do B. T., Chang H. Y., et al. (2016) irCLIP platform for efficient characterization of protein-RNA interactions. Nat Methods 13: 489-492
• 1. Martin G. and Zavolan M. (2016) Redesigning CLIP for efficiency, accuracy and speed. Nat Meth 13: 482-483

1. Martin G. and Zavolan M. Redesigning CLIP for efficiency, accuracy and speed. Nat Methods. 2016;13:482-483
2. Haque N. and Hogg J. R. Easier, Better, Faster, Stronger: Improved Methods for RNA-Protein Interaction Studies. Mol Cell. 2016;62:650-651

1. Zarnegar B. J., Flynn R. A., Shen Y., Do B. T., Chang H. Y. and Khavari P. A. irCLIP platform for efficient characterization of protein-RNA interactions. Nat Methods. 2016;13:489-492