TITLE: Generation of Library for Sequencing
AUTHORS: Clémentine Delan-Forino, David Tollervey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The RNA exosome complex functions in both the accurate processing and rapid degradation of many classes of RNA in eukaryotes and Archaea. Functional and structural analyses indicate that RNA can either be threaded through the central channel of the exosome or more directly access the active sites of the ribonucleases Rrp44 and Rrp6, but in most cases, it remains unclear how many substrates follow each pathway in vivo. Here we describe the method for using an UV cross-linking technique termed CRAC to generate stringent, transcriptome-wide mapping of exosome–substrate interaction sites in vivo and at base-pair resolution.</div><br/><div class = "text-block"><span>We present a protocol for the identification of RNA interaction sites for the exosome, using UV cross-linking and analysis of </span><span style = "font-style:italic;">c</span><span>DNA (CRAC) [</span><span style = ":UNDERLINE;">1</span><span>, </span><span style = ":UNDERLINE;">2</span><span>]. A number of related protocols for the identification of sites of RNA–protein interaction have been reported, including HITS-CLIP, CLIP-Seq, iCLIP, eCLIP, and others [</span><span style = ":UNDERLINE;">3</span><span>, </span><span style = ":UNDERLINE;">4</span><span>, </span><span style = ":UNDERLINE;">5</span><span>, </span><span style = ":UNDERLINE;">6</span><span>]. These all exploit protein immunoprecipitation to isolate protein–RNA complexes. CRAC is distinguished by the inclusion of tandem affinity purification and denaturing purification, allowing greater stringency in the recovery of authentic RNA–protein interaction sites.</span></div><br/><div class = "text-block"><span>To allow CRAC analyses, strains are created that express a “bait” protein with a tripartite tag. This generally consists of His6, followed by a TEV-protease cleavage site, then two copies of the z-domain from Protein A (HTP). The tag is inserted at the C terminus of the endogenous gene within the chromosome. The fusion construct is the only version of the protein expressed and this is under the control of the endogenous promoter. Several alternative tags have been successfully used, including a version with N-terminal fusion to a tag consisting of 3× FLAG-PreSission protease (PP) cleavage site-His6 (FPH) [</span><span style = ":UNDERLINE;">7</span><span>]. This is a smaller construct and is suitable for use on proteins with structures that are incompatible with C-terminal tagging. An additional variant is the insertion of a PP site into a protein that is also HTP tagged. This allows the separation of different domains of multidomain proteins. Importantly, the intact protein is cross-linked in the living cell, with domain separation in vitro. This has been successfully applied to the exosome subunit Rrp44/Dis3 to specifically identify binding sites for the PIN endonuclease domain [</span><span style = ":UNDERLINE;">8</span><span>].</span></div><br/><div class = "text-block"><span>Briefly, during standard CRAC analyses, covalently linked protein–exosome complexes are generated in vivo by irradiation with UV-C (254 nm). This generates RNA radicals that rapidly react with proteins in direct contact with the affected nucleotide (zero length cross-linking). The cells are then lysed and complexes with the bait protein are purified using an IgG column. Protein–RNA complexes are specifically eluted by TEV cleavage of the fusion protein and cross-linked RNAs trimmed using RNase A/T1, leaving a protected “footprint” of the protein binding site on the RNA. Trimmed complexes are denatured using 6 M Guanidinium, immobilized on Ni-NTA affinity resin and washed under denaturing conditions to dissociate copurifying proteins and complexes. The subsequent enzymatic steps are all performed on-column, during which RNA 3′ and 5′ ends are prepared, labeled with </span><span style = "vertical-align:super;">32</span><span>P (to allow RNA–protein complexes to be followed during gel separation) and linkers ligated. Note, however, that alternatives to using </span><span style = "vertical-align:super;">32</span><span>P labeling have been reported (e.g., [</span><span style = ":UNDERLINE;">6</span><span>]). The linker-ligated, RNA–protein complexes are eluted from the Ni-NTA resin and size selected on a denaturing SDS-PAGE gel. Following elution, the bound RNA is released by degradation of the bait protein using treatment with Proteinase K. The recovered RNA fragments are identified by reverse transcription, PCR amplification and sequencing using an Illumina platform.</span></div><br/><div class = "text-block"><span>Relative to CLIP-related protocols, CRAC offers the advantages of stringent purification, that substantially reduces background, and on-bead linker ligation that simplifies separation of reaction constituents during successive enzymatic steps. It also avoids the necessity to generate high-affinity antibodies needed for immunoprecipitation. Potential disadvantages are that, despite their ubiquitous use in yeast studies, tagged constructs may not be fully functional. This can be partially mitigated by confirming the ability of the tagged protein to support normal cell growth and/or RNA processing, or by comparing the behavior of N- and C-terminal tagged constructs. Additionally, because linkers are ligated to the protein–RNA complex, a possible disadvantage is that UV-cross-linking of the RNA at, or near, the 5′ or 3′ end it may sterically hinder on-column (de)phosphorylation and/or linker ligation. With these caveats, CRAC has been successfully applied to >50 proteins in budding yeast, and in other systems ranging from pathogenic bacteria to viral infected mouse cells [</span><span style = ":UNDERLINE;">7</span><span>, </span><span style = ":UNDERLINE;">9</span><span>].</span></div></div>

[STEPS]
?. [Reverse Transcription of Purified RNA]
To increase the efficiency of this step, prepare fresh dNTP dilution prior RT or aliquot and store at   to avoid multiple thawing.
?. [PCR Amplification of cDNA Libraries]
The number of cycles used to prepare cDNA libraries should be optimized for the template and limited to minimize artifacts due to overamplification, that is, the frequency of PCR duplicates. Generally, 21–22 cycles have been sufficient to produce complex libraries from cDNA generated from Exosome subunit-bound RNA, however we typically vary between 19 and 24 cycles and will increase number of independent PCR reactions (up to 5) for samples with low abundance of cDNA.
?. [Size Selection of cDNA Libraries on Gel]
At this stage, it is possible to adjust library size distribution and enrich the DNA library for cDNA of a certain length before sequencing. This size selection is dependent on the length of sequencing that will be used, the protein, and the biological questions CRAC is supposed to answer. If 50 bp sequencing length is planned, it is not useful to recover extra-long cDNAs; moreover longer sequences will decrease resolution of protein binding sites. On the other hand, for most proteins, it is preferable to avoid overpopulation of the library by short sequences (shorter than 20 nt), which are difficult to map confidently. In some case, these general guidelines have to be adjusted for biological relevance: for instance, cDNA libraries from Rrp44-HTP are cut just above 130 nt to also recover short sequences enriched in cDNAs corresponding to RNAs bypassing the long exosome channel and directly accessing Rrp44.
?. [Sequencing]
The samples can be submitted for single end sequencing on Illumina MiSeq, HiSeq, MiniSeq, or NextSeq platforms.The read depth required for sufficient coverage of binding sites will depend on the number of RBP binding sites and complexity of the library generated (i.e., number of PCR duplicates). The exosome binds a huge diversity of targets. Since the highest proportion of the reads are aligned to ribosomal RNA, it is necessary to sequence deeply enough to detect less frequently bound targets. We generally aim to generate 17–35 nt trimmed RNA fragments that contain enough sequence information for a unique alignment, and that are short enough to ensure the protein interaction site is contained within the sequenced portion. We routinely use Illumina 50 bp single end sequencing, which is long enough to sequence into the 3′ adapter sequence.
?. [Reverse Transcription of Purified RNA]
Resuspend the RNA pellet in . Add  and .
[MilliQ water]
[RT primer [10 μM]]
[10 mM dNTPs]
?. [Reverse Transcription of Purified RNA]
Heat the samples to  for , then chill  for . Collect the contents by brief centrifugation.
80 °C
on ice
?. [Reverse Transcription of Purified RNA]
To each sample, add  (Invitrogen), , and .
[5× First Strand buffer]
[100 mM DTT]
[RNasin]
?. [Reverse Transcription of Purified RNA]
Incubate at  for  and add  (Invitrogen). This step will help dissociate any nonspecifically annealed primers from the RNA.
50 °C
[SuperScript III]
?. [Reverse Transcription of Purified RNA]
Incubate at  for .
50 °C
?. [Reverse Transcription of Purified RNA]
Inactivate the Superscript III by incubating the samples at  for .
65 °C
?. [Reverse Transcription of Purified RNA]
Add  and incubate for  at .
[RNase H]
37 °C
?. [PCR Amplification of cDNA Libraries]
To , add  containing:
[cDNA template]
[PCR master mix]
[10× LA Taq buffer]
[10 μM P5 Solexa primer]
[10 μM pE_miRCat reverse primer]
[(fresh) 10 mM dNTPs]
[ LA TaKaRa Taq polymerase]
[nuclease-free water]
We prepare three or more PCR reactions per sample to increase the complexity of our libraries.
?. [PCR Amplification of cDNA Libraries]
The reaction is run with the following cycling conditions:  ABC1TempTimeCycle295&nbsp;°C2&nbsp;min&nbsp;398&nbsp;°C20&nbsp;s21&nbsp;cycles452&nbsp;°C20&nbsp;s568&nbsp;°C20&nbsp;s672&nbsp;°C5&nbsp;min&nbsp;
ABC1TempTimeCycle295&nbsp;°C2&nbsp;min&nbsp;398&nbsp;°C20&nbsp;s21&nbsp;cycles452&nbsp;°C20&nbsp;s568&nbsp;°C20&nbsp;s672&nbsp;°C5&nbsp;min&nbsp;
?. [PCR Amplification of cDNA Libraries]
Pool PCR reactions into a clean microcentrifuge tube and precipitate with 0.1 volume sodium acetate (pH 5.2) and 2.5 volumes of ice cold absolute ethanol.
Alternatively, you can concentrate cDNA libraries using MinElute PCR purification kit as indicated in the manufacturer’s instructions. Elute your samples with  .
?. [PCR Amplification of cDNA Libraries]
Incubate at  for  (it is better to not precipitate longer to avoid recovering too much salt). Centrifuge at .
-20 °C
Centrifuge: 16000 34, 30 min, 4 10
?. [PCR Amplification of cDNA Libraries]
Remove the supernatant and air dry the pellet. Resuspend in .
[MilliQ water]
?. [Size Selection of cDNA Libraries on Gel]
Prepare a 3% Metaphor agarose gel using 1× TBE buffer (with 1:1000 SYBR Safe) and store it at  for a minimum of .
4 °C
Preparing a Metaphor gel takes longer than preparing a standard agarose gel, and it is common for the agarose to form “lumps” which are hard to dissolve. One option is to let the Metaphor powder to soak for 30 min in 1× TBE before agitating it on a magnetic stirrer hot plate. A second option is to microwave the mixture before agitating it on a magnetic stirrer hot plate. The gel can be prepared the day before and stored at 4 °C wrapped in cling film.
?. [Size Selection of cDNA Libraries on Gel]
Add  to precipitated sample and load the entire volume onto the prepared 3% Metaphor agarose gel along with 50 bp DNA ladder.
[6× DNA gel loading dye]
?. [Size Selection of cDNA Libraries on Gel]
Run the gel at 80 V for approximately  or until the bromophenol blue dye front reaches 2 cm from the edge of the gel.
?. [Size Selection of cDNA Libraries on Gel]
Image the gel.
We use a Typhoon FLA9500 laser scanner (GE Life sciences) for increased sensitivity and print the gel images at 1:1 scale. A lower band around 120 nt corresponding to the amplified sequencing adapter dimers is sometimes visible and should be avoided when cutting. The cDNA libraries appear as a smear running above primer dimers that should be apparent in the negative control samples. The presence of a sharp band may indicate excessive RNA digestion. For other proteins, this can simply indicate the presence of a highly abundant binding target. However, this has not been observed with exosome components. Lack, or small amounts, of PCR products on the agarose gel (despite strong signal by autoradiography) suggests inefficient enzymatic reactions.
?. [Size Selection of cDNA Libraries on Gel]
Place the gel on a transparent film and align it to the 1:1 scan of the gel. Excise the libraries using a sterile scalpel by cutting from the bottom of the smear to the predefined upper limit.
?. [Size Selection of cDNA Libraries on Gel]
Transfer the gel slices to 2 ml microcentrifuge tubes. Rescan gel afterward to check the expected bits are cut out.
?. [Size Selection of cDNA Libraries on Gel]
Add  from the MinElute Gel Extraction purification kit (QIAgen) and incubate the gel slices at  for – to dissolve the agarose.
[Buffer QG]
42 °C
?. [Size Selection of cDNA Libraries on Gel]
Transfer the volume to a MinElute column fitted to collection tubes and spin at . Discard the flowthrough. Repeat with the leftover of buffer/agarose to bind all the sample to the same column.
Centrifuge: 16000 34, 1 min
?. [Size Selection of cDNA Libraries on Gel]
Add  and spin at . Discard the flowthrough.
[Buffer QG]
Centrifuge: 16000 34, 1 min
?. [Size Selection of cDNA Libraries on Gel]
Add  (QIAgen) to the columns and incubate for  at . Spin at  and discard the flowthrough.
[ Buffer PE]
0 Room temperature
Centrifuge: 16000 34, 1 min
?. [Size Selection of cDNA Libraries on Gel]
Dry the columns by spinning at . Transfer the columns to clean 1.5 ml microcentrifuge tubes.
Centrifuge: 16000 34, 2 min
?. [Size Selection of cDNA Libraries on Gel]
Add  on membrane and let stand for –. Elute the purified cDNA by spinning at .
[MilliQ water]
Centrifuge: 16000 34, 1 min
?. [Size Selection of cDNA Libraries on Gel]
Quantify the cDNA library using a Qubit high sensitivity DNA assay kit and fluorometer and store the libraries at .
-20 °C