CLIP with BrdU Affinity Purification
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 (Weyn-Vanhentenryck et al., 2014). 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.
- 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
- Tube-column transfers can result in major loss of material (Zarnegar et al., 2016)
- Not yet adopted widely by the scientific community
Illumina Library prep and Array Kit Selector
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