iCLIP identifies protein-RNA interactions
by UV cross-linking of intact cells or tissue
UV
UV
UV
Immunoprecipitation
and RNA ligation
SDS-PAGE and
proteinase treatment
Reverse transcription
cDNA circularisation
Linearisation, PCR
and high-throughput
sequencing
Individual-nucleotide resolution
cross-linking and immunoprecipitation
(iCLIP) determines the genome-wide
profile of RNA sites bound by an RBP
(Konig et al, Nat Rev Genet, 2012).
The technique employs ultraviolet light
to form a covalent bond between RBPs
(orange oval) and RNA molecules. The
protein-RNA complex is purified and
ligated to an adapter, radiolabelled and
visualized, and the RNAs are released
upon digestion of the protein. The
primers for reverse transcription contain unique barcodes (marked in red),
which enable library multiplexing, and
removal of the artifacts of PCR amplification. This enables to prepare a cDNA
library for quantitative analysis of
protein-RNA interactions via
high-throughput sequencing.