Abstract
Crosslinking and immunoprecipitation (CLIP) methods are powerful techniques to interrogate direct protein-RNA interactions and dissect posttranscriptional gene regulatory networks. One widely used CLIP variant is photoactivatable ribonucleoside enhanced CLIP (PAR-CLIP) that involves in vivo labeling of nascent RNAs with the photoreactive nucleosides 4-thiouridine (4SU) or 6-thioguanosine (6SG), which can efficiently crosslink to interacting proteins using UVA and UVB light. Crosslinking of 4SU or 6SG to interacting amino acids changes their base-pairing properties and results in characteristic mutations in cDNA libraries prepared for high-throughput sequencing, which can be computationally exploited to remove abundant background from non-crosslinked sequences and help pinpoint RNA binding protein binding sites at nucleotide resolution on a transcriptome-wide scale. Here we present a streamlined protocol for fluorescence-based PAR-CLIP (fPAR-CLIP) that eliminates the need to use radioactivity. It is based on direct ligation of a fluorescently labeled adapter to the 3'end of crosslinked RNA on immobilized ribonucleoproteins, followed by isolation of the adapter-ligated RNA and efficient conversion into cDNA without the previously needed size fractionation on denaturing polyacrylamide gels. These improvements cut the experimentation by half to 2 days and increases sensitivity by 10-100-fold.