that may be of interest, allow accurate isolation of RNAs that are of
suitable length for library construction, and to provide an addi-
tional purification of free RNA that may stick to the beads used in
the immunoprecipitation.
The iCLIP protocol has been extensively optimized in recent
years, and we refer the reader to two closely related publications
from the Ule [13] and Konig [29] research groups that will assistTable 1
(continued)
Method Difference Advantage Disadvantages References
CRAC Dual purification
through use of
cleavable bipartite
tag that can allow
purification
without
antibodiesAllows analysis of RBPs
with no/poor
antibodies for iCLIP,
purify RBP–RNA
complexes under
highly denaturing
conditionsRequires RBP tagging
so not endogenous
(i.e., functional
testing of tagged
protein required)[23]eCLIP Modified iCLIP
workflow
includes 3^0
adapter ligated to
cDNA to avoid
circularization
step, and no
visualization of
the purified
protein–RNA
complexReduced protocol
duration and
improved efficiency
of ligation steps
allows high-
throughput analysis
of many RBPsNo complex
visualization can
mean non-antigen
contamination of
library. Blind cut
means library may be
dominated by short
RNAs around the
antigen Mw that may
reduce efficiency in
protocol[24]Fast-iCLIP Modified library
preparation to
reduce iCLIP
protocol durationQuick biotin
purification of
CLIP’d material
instead of
precipitations,
improved efficiency
of circLigase stepInsert size selection
only post-PCR may
bias library toward
short products[25]irCLIP Further
optimization of
fast-iCLIP
protocol and
introduction of
an adapter for
non-radioactive
visualization of
the purified
protein–RNA
complexNear-infrared adapter is
as sensitive as
radioactive labeling,
dot blots allow
monitoring of RNA
inputs into library
preparation steps,
increased recovery of
protein–RNA
complexes from
membrane, less tube
transfersLess stringent size
selection of cDNA
inserts due to bead-
based cleanups and
PCR[26]430 Christopher R. Sibley