The 5 mM sodium m-periodate and binding buffer must always
be made fresh as the former is light sensitive and the latter contains
protein lysates. The other buffers can be stored longer, but the use
of fresh buffer is always advised whenever possible.
2.4 Miscellaneous
1. 2 mL Eppendorf tubes (2 Â number of reactions).
2. 1.5 mL Eppendorf tubes (2 Â number of reactions).
3. 15 mL Falcon tubes (1 Â number of reactions).
4. 50 mL Falcon tubes or other sterilized containers for storing
buffers.
3 Methods
3.1 Designing Probe
Sequences
Since RBPs can bind to their target RNAs in a sequence-specific
manner, RNA sequences used as probes in the protocol should be
significantly enriched at suspected RBP-binding sites as those
sequences are likely to correspond to the binding motif of an
RBP. To identify binding sites, users can use techniques such as
gCLIP-seq [8] or PIP-seq [9]. gCLIP-seq identifies the binding
sites of RBPs throughout the genome based on protein-RNA crosslinking resulting in mutations and truncated read ends in the
resulting high-throughput sequencing data. PIP-seq is an
antibody-independent sequencing technique that uses RNase
digestion in the presence compared to the absence of bound
RBPs to identify transcriptome-wide RBP-binding sites. Thus,
both protocols allow researchers to unbiasedly survey
RBP-binding sites on a transcriptome-wide scale. However,
researchers will not be able to identify the RBPs that bind to each
identified RBP-bound site based simply on the data from these
approaches alone. From the total collection of identified
RBP-binding sites, users can identify enriched sequences using
motif enrichment analysis tools such as MEME [12] or HOMER
[13] (Fig. 2) (see Note 6).
Figure 2 provides a general overview of motif enrichment and
selection. In more detail, both MEME and HOMER operate under
the same general principle of scanning through a collection of
sequences and identifying consensus sequences that occur multiple
times. As part of that process, the algorithm will compare sequence
representation against a background distribution of all possible
n-mers. While both algorithms provide a default background
model as a starting point, users can also supply their own custom
background sequences to better fine-tune results. For example,
users can create organism-specific background models or classify
sequences of interest into untranslated regions (UTRs), CDS,
introns, etc. and then supply the relevant custom background.
This has the advantage of correcting for species-specific biases in
Using Bead-Tagged RNAs to Identify RBPs
245
be made fresh as the former is light sensitive and the latter contains
protein lysates. The other buffers can be stored longer, but the use
of fresh buffer is always advised whenever possible.
2.4 Miscellaneous
1. 2 mL Eppendorf tubes (2 Â number of reactions).
2. 1.5 mL Eppendorf tubes (2 Â number of reactions).
3. 15 mL Falcon tubes (1 Â number of reactions).
4. 50 mL Falcon tubes or other sterilized containers for storing
buffers.
3 Methods
3.1 Designing Probe
Sequences
Since RBPs can bind to their target RNAs in a sequence-specific
manner, RNA sequences used as probes in the protocol should be
significantly enriched at suspected RBP-binding sites as those
sequences are likely to correspond to the binding motif of an
RBP. To identify binding sites, users can use techniques such as
gCLIP-seq [8] or PIP-seq [9]. gCLIP-seq identifies the binding
sites of RBPs throughout the genome based on protein-RNA crosslinking resulting in mutations and truncated read ends in the
resulting high-throughput sequencing data. PIP-seq is an
antibody-independent sequencing technique that uses RNase
digestion in the presence compared to the absence of bound
RBPs to identify transcriptome-wide RBP-binding sites. Thus,
both protocols allow researchers to unbiasedly survey
RBP-binding sites on a transcriptome-wide scale. However,
researchers will not be able to identify the RBPs that bind to each
identified RBP-bound site based simply on the data from these
approaches alone. From the total collection of identified
RBP-binding sites, users can identify enriched sequences using
motif enrichment analysis tools such as MEME [12] or HOMER
[13] (Fig. 2) (see Note 6).
Figure 2 provides a general overview of motif enrichment and
selection. In more detail, both MEME and HOMER operate under
the same general principle of scanning through a collection of
sequences and identifying consensus sequences that occur multiple
times. As part of that process, the algorithm will compare sequence
representation against a background distribution of all possible
n-mers. While both algorithms provide a default background
model as a starting point, users can also supply their own custom
background sequences to better fine-tune results. For example,
users can create organism-specific background models or classify
sequences of interest into untranslated regions (UTRs), CDS,
introns, etc. and then supply the relevant custom background.
This has the advantage of correcting for species-specific biases in
Using Bead-Tagged RNAs to Identify RBPs
245