RNA Detection

aatgatacggcgaccaccgagatctacactctttcccta

CACGACGCTCTTCCGATCT

gtccttacggctctggctagagcatacgg

cagaagacgaac

TCTAGCCTTCTCGCCAAGTC

RBP

254nM

UV

UV cross-

linking in vivo

5’ RBP 3’ 5’ RBP 3’+

Immunoprecipitation

of RBP-of-interest Adapter ligation

-Ab

RNase

-UV

Excised

membrane

H

ML

cDNA size

selection

Reverse

transcription

Protein-RNA complex

visualisation & excision

High Throughput

Sequencing

Lysis and

RNase I

digestion

3’ end

dephosphorylation

SDS-PAGE

Proteinase K

& RNA

extraction

RNA

alkaline

hydrolysis

Crosslinked

peptide

BamH1

cutting

cDNA

circularisation

GATCC

ACGACGCTCTTCaaaa

GTTCAG

CGCCAAGTCCTAGG

TGCTGCGAGAAGGCTAGAnnnccaann

....RNA...

...cDNA..............

AGATCGGAAGAGCGGTTCAGaaaaaaaaaaaa/iAzideN/aaaaaaaaaaaa/Bio/tcccc

Circularised cDNA

(Step 3.10.6)

= L3-IR-app (Step 3.5.8)

= RT primer (Step 3.8.6)

= Cut4oligo (Step 3.10.7)

= P5/P3 Solexa primers (Steps 3.11.6 / 12 /18)

GGATCCCCTAGG= BamH1 cut site (Step 3.10.9)

lowercase= Unaligned nucleotides

A.

B.

= =

Fig. 1The iCLIP protocol. (a) The iCLIP protocol begins by cross-linking samples in order to covalently bind
RBPs and their RNA targets together. Samples are then lysed before a controlled RNase I digestion is carried
out in order to shorten cross-linked RNA fragments to lengths compatible with RNA sequencing. At this point

iCLIP to Determine Protein-RNA Interactions 431