BrdU-CLIP

BrdU-CLIP

BrdU-CLIP sequences the binding sites of RBPs with single-nucleotide resolution. BrdU-CLIP fixes a major problem in HITS-CLIP where 5' adapters are not attached to the cDNA due to premature termination of reverse transcription. BrdU-CLIP uses BrdUTPs and primers with 3' and 5' adapters during reverse transcription. This method enables greater cDNA yield and attaches both 5' and 3' adapters in a single step.

First, RNA-protein complexes are UV-crosslinked, digested with RNase, and immunoprecipitated. 3' adapters are ligated to the RNA and reverse-crosslinked by proteinase K digestion. Some peptides from the crosslink remain bonded with the RNA strands, even after reverse-crosslinkage. RT is carried out using BrdUTPs and primers containing 3' and 5' adapters separated by an apurinic/apyrimidinic endonuclease (APE) cleavage site. The resultant cDNA is purified, circularized, and further purified by BrdU pull-down. Next, the cDNA is linearized by cleaving at the APE site, PCR-amplified, and sequenced.

Pros:

• Sequences RNA-binding sites of RBPs with single-nucleotide resolution
• Captures both truncated and nontruncated cDNAs, unlike HITS-CLIP
• Provides higher cDNA yield due to BrdUTP
• Attaches 3' and 5' adapters in one RT step

Cons:

• Tube-column transfers can result in major loss of material¹
• Not yet adopted widely by the scientific community

• BrdU-CLIP: Weyn-Vanhentenryck S. M., Mele A., Yan Q., Sun S., Farny N., et al. (2014) HITS-CLIP and integrative modeling define the Rbfox splicing-regulatory network linked to brain development and autism. Cell Rep 6: 1139-1152
• 1. 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

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2. Vuong C. K., Black D. L. and Zheng S. The neurogenetics of alternative splicing. Nat Rev Neurosci. 2016;17:265-281
3. Hsiao Y.-H. E., Cass A. A., Bahn J. H., Lin X. and Xiao X. Global Approaches to Alternative Splicing and Its Regulation—Recent Advances and Open Questions. Transcriptomics and Gene Regulation. 2016;37-71
4. Bangru S. and Kalsotra A. Advances in analyzing RNA diversity in eukaryotic transcriptomes: peering through the Omics lens. F1000Research. 2016;5:2668
5. Flynn R. A., Martin L., Spitale R. C., Do B. T., Sagan S. M., et al. Dissecting noncoding and pathogen RNA-protein interactomes. RNA. 2015;21:135-143

1. Weyn-Vanhentenryck S. M., Mele A., Yan Q., Sun S., Farny N., et al. (2014) HITS-CLIP and integrative modeling define the Rbfox splicing-regulatory network linked to brain development and autism. Cell Rep 6: 1139-1152