Grant support is from the National Basic Research Program of China (2017YFA0504204, 2018YFA0107604), the National Natural Science Foundation of China (31630095), and the Center for Life Sciences at Tsinghua University.

1. Cell line construction
<ol>
	<li>Clone the gene of interest into a PiggyBac vector pPiggyBac-FLAG-bio-[cDNA of interest]-(Hygromycin-resistant) plasmid that carries a FLAG-biotin epitope13,21 to express a FLAG-biotin tag fused RBP (FBRBP).</li>
	<li>Cotransfect the FBRBP expressing vector with the pBase vector into a cell line expressing the BirA enzyme22. 
	NOTE: In this study, the FBRBP gene was transfected into mouse embryonic stem cells (mESCs) carrying an integrated BirA expression vector21. Alternatively, the FBRBP expressing vector can be cotransfected into cells with pBase and pPiggyBac-BirA-V5 together.</li>
	<li>Grow the cells in mESC on gelatinized dishes and maintain in mES culture medium (Table of Materials) in 5% CO2 at 37 &#176;C. Select the transfected cells with hygromycin b (100 &#181;g/mL) for 1 week.</li>
	<li>After drug selection, harvest cells by SDS loading buffer (Table 1) and use a Western blot23 to confirm the tagging and expression of the gene with a FLAG antibody and streptavidin-HRP, respectively.</li>
</ol>
2. Cross-linking
<ol>
	<li>Plate ~3 x 106 mES cells in a 10 cm plate 1 day before the experiment so that the cells will grow to 70&#8722;90% confluence when harvested (~5&#8722;10 million cells per sample).</li>
	<li>Remove the medium with a vacuum. Treat the cells with 2 mL of 0.25% trypsin-EDTA for 2 min at room temperature (RT) and quench the trypsin by 4 mL of fresh mESC medium. Transfer the cells to a 15 mL centrifuge tube. Spin down the cells by centrifugation at 300 x g for 3 min at 4 &#176;C and remove the supernatant.</li>
	<li>Suspend the cells with 4 mL of cold PBS and plate the cells back to the 10 cm plate for cross-linking. Cross-link the cells by radiation with 254 nm UV light (set the UV cross-linker with a parameter of 400 mJ/cm2). 
	NOTE: For cell monolayers, the cells can be cross-linked with UV light directly on the plate before trypsin treatment.</li>
	<li>Transfer the cross-linked cells to a 15 mL tube. Spin down by centrifugation at 300 x g for 3 min at 4 &#176;C and remove the supernatant. 
	NOTE: The cross-linked cell pellet can be stored at -80 &#176;C until use.</li>
</ol>
3. Cell lysate preparation
<ol>
	<li>Lyse the cells with 500 &#181;L of wash buffer A (Table 1) freshly supplemented with 1 mM DTT, 1 mM PMSF, 1/500 protease inhibitor cocktail, and 400 U/mL RNase inhibitor. Transfer the cells to an RNase-free 1.5 mL tube. Incubate the cells for 30&#8722;60 min on a rotor with gentle rotation at 4 &#176;C.</li>
	<li>Treat the lysate with 30 &#181;L of DNase I at 37 &#176;C for 10 min. Stop the reaction by adding 4 &#181;L of 0.5 M EDTA. Spin down the insoluble pellet by 12,000 x g for 20 min at 4 &#176;C and transfer the supernatant to a new prechilled 1.5 mL tube. 
	NOTE: For immediate use, keep on ice. If the supernatant will not be used immediately, store it at -80 &#176;C until use.</li>
</ol>
4. FLAG beads preparation
<ol>
	<li>Add 40 &#181;L of slurry FLAG beads per sample to a fresh 1.5 mL tube.</li>
	<li>Quickly wash the beads with 0.5 mL of ice-cold wash buffer A and spin down by 3,000 x g for 2 min at 4 &#176;C.</li>
	<li>Repeat step 4.2 once. Spin down the beads with 3,000 x g for 2 min at 4 &#176;C. Carefully remove the buffer with a narrow-end pipette tip. Avoid removing the beads.</li>
</ol>
5. Immunoprecipitation
<ol>
	<li>Transfer the cell lysate from step 3.2 to the pre-equilibrated FLAG beads. Rotate all the samples on a roller shaker at 4 &#176;C gently. Incubate the FLAG beads with lysate for 2&#8722;4 h or overnight.</li>
	<li>Centrifuge the beads for 2 min at 3,000 x g and remove the supernatant.</li>
	<li>Wash the beads with 0.5 mL of prechilled wash buffer A by incubation for 5 min in a rotator at 4 &#176;C, spin down the beads with 3,000 x g for 2 min and remove the supernatant. Repeat the wash 1x.</li>
	<li>Repeat the wash 2x as described in step 5.3 with 0.5 mL of prechilled wash buffer B (Table 1).</li>
</ol>
6. Elution with 3x FLAG peptide
<ol>
	<li>Prepare 3x FLAG elution solution by dissolving 1 mg of 3x FLAG peptide with 5 mL of wash buffer A to a final concentration of 200 ng/mL.</li>
	<li>Spin down the FLAG beads from step 5.4 with 3,000 x g for 2 min. Remove the supernatant carefully. Make sure most of the supernatant is removed by using a narrow end pipette tip.</li>
	<li>Add 200 &#181;L of 3x FLAG elution solution to each sample. Incubate the samples with gentle rotation for 30 min at 4 &#176;C. Spin down the FLAG beads for 2 min at 3,000 x g. Transfer the supernatants to fresh tubes.</li>
	<li>Repeat the elution 2x as described in steps 6.2 and 6.3 and pool the eluents together. Save 5% of the eluents for Western blot analysis or silver staining to check the efficiency of FLAG-IP.</li>
</ol>
7. Streptavidin beads preparation
<ol>
	<li>Prepare 50 &#181;L of a streptavidin bead slurry for each sample. Collect the beads with a magnetic stand for 30 s and remove the supernatant with a pipette tip.</li>
	<li>Quickly wash the beads with 0.5 mL of ice-cold wash buffer A and collect the beads with a magnetic stand for 30 s.</li>
	<li>Repeat the wash 1x. Remove the supernatant after the wash.</li>
</ol>
8. Biotin affinity purification
<ol>
	<li>Transfer the pooled eluents from step 6.4 to the streptavidin beads from step 7.3 and gently rotate the samples at 4 &#176;C for 1&#8722;3 h or overnight.</li>
	<li>Collect the beads with a magnetic stand for 30 s and remove the supernatant. Wash the beads 2x with 500 &#181;L of wash buffer C (Table 1) by gentle rotation at RT for 5 min.</li>
	<li>Collect the beads with the magnetic stand and remove the supernatant. Wash the beads 2x with 500 &#181;L of wash buffer D (Table 1) by rotating at RT for 5 min.</li>
	<li>Collect the beads with the magnetic stand and remove the supernatant. Quickly wash the beads 2x with 500 &#181;L of ice-cold PNK buffer (Table 1).</li>
</ol>
9. Partial RNA digestion
<ol>
	<li>Make a 105-fold dilution of MNase with MNase reaction buffer (Table 1). Add 100 &#181;L of MNase solution to each sample. Vortex the beads at 37 &#176;C with a thermal mixer set at 1,200 rpm (5 s run, 30 s stop) for 10 min, collect the beads with a magnetic stand for 30 s and remove the supernatant. 
	NOTE: The concentration of MNase should be optimized for different RNA-binding proteins. The MNase concentration that can digest RNAs into 30&#8722;50 nt fragments (determined by the size of insert of the final library) is optimal.</li>
	<li>Quickly wash the beads 2x with 500 &#181;L of ice-cold PNK+EGTA buffer (Table 1). Collect the beads with a magnetic stand and remove the supernatant between each wash step.</li>
	<li>Wash the beads 2x with 500 &#181;L of wash buffer C by gentle rotation for 5 min at RT. Then quickly wash the beads 2x with 500 &#181;L of ice-cold PNK buffer.</li>
</ol>
10. Dephosphorylation of RNA
<ol>
	<li>Make a calf intestine phosphatase (CIP) reaction mix with 8 &#181;L of 10x CIP buffer (Table of Materials), 3 &#181;L of CIP enzyme, and 69 &#181;L of water. The total volume is 80 &#181;L per reaction.</li>
	<li>Collect the beads with a magnetic stand and remove the buffer. Add the CIP reaction mix to the beads. Vortex the beads at 37 &#176;C with a thermal mixer set at 1,200 rpm (5 s run, 30 s stop) for 10 min.</li>
	<li>Quickly wash the beads 2x with 500 &#181;L of ice-cold PNK+EGTA buffer. Quickly wash the beads 2x with 500 &#181;L of ice-cold PNK buffer.</li>
</ol>
11. 3&#8217; linker ligation
<ol>
	<li>Make a 3&#8217; linker mix with 4 &#181;L of 20 &#181;M 3&#8217; linker, 4 &#181;L of 10x T4 RNA ligase buffer, 4 &#181;L of 50% PEG8000, 2 &#181;L of T4 RNA ligase 2 (truncated), and 26 &#181;L of water. The total volume is 40 &#181;L per reaction.</li>
	<li>Collect the beads from step 10.3 with a magnetic stand and remove the buffer. Add 40 &#181;L of the 3&#8217; linker ligation mix to the beads. Vortex the beads at 16 &#176;C with a thermal mixer set at 1,200 rpm (5 s run, 30 s stop) for 3 h or overnight.</li>
	<li>Quickly wash the beads 2x with 500 &#181;L of ice-cold PNK+EGTA buffer. Quickly wash the beads 2x with 500 &#181;L of ice-cold PNK buffer.</li>
</ol>
12. PNK treatment
<ol>
	<li>Make a PNK mix with 4 &#181;L of 10x PNK buffer, 2 &#181;L of T4 PNK enzyme, 1 &#181;L of 10 mM ATP, and 33 &#181;L of water. The total volume is 40 &#181;L per reaction.</li>
	<li>Collect the beads with a magnetic stand and remove the buffer. Add 40 &#181;L of PNK mix to the beads. Vortex the beads gently at 37 &#176;C with a thermal mixer set at 1,200 rpm (5 s run, 30 s stop) for 10 min.</li>
	<li>Quickly wash the beads with 500 &#181;L of ice-cold PNK+EGTA buffer.</li>
	<li>Quickly wash the beads with 500 &#181;L of ice-cold PNK buffer. Save 5% of the beads for Western or silver staining analysis to confirm the efficiency of immunoprecipitation.</li>
</ol>
13. RNA isolation
<ol>
	<li>Collect the beads from step 12.4 with a magnetic stand and remove the buffer. Add 200 &#181;L of proteinase K digestion buffer (Table 1). Vortex the beads for 30 min at 37 &#176;C with a thermal mixer set at 1,200 rpm (5 s run, 30 s stop). Magnetically isolate the beads and take the supernatant for the experiment.</li>
	<li>Add 400 &#181;L of RNA isolation reagent (Table of Materials) to the supernatant from step 13.1, mix thoroughly, and incubate on ice for 5 min. Add 80 &#181;L of chloroform and mix thoroughly and then incubate on ice for 5 min. Spin down with 12,000 x g for 10 min 4 &#176;C. 
	NOTE: This step should be performed in a chemical hood.</li>
	<li>Take the supernatant (~500 &#181;L) and add two volumes of isopropanol:ethanol mix (1:1), 1/10 volume of 3 M sodium acetate (pH = 5.5), and 1 &#181;L of glycogen. Freeze at -20 &#176;C for 2 h. Centrifuge at 4 &#176;C with 12,000 x g for 20 min.</li>
	<li>Wash the pellet 2x with ice-cold 70% ethanol. Spin down the pellet at 4 &#176;C with 12,000 x g for 5 min. Remove the supernatant and dry the pellet. Dissolve the RNAs in 5 &#181;L of RNase-free H2O.</li>
</ol>
14. 5&#8217; RNA linker ligation
<ol>
	<li>Make a 5&#8217; RNA ligation mix with 1 &#181;L of 10x T4 RNA ligase buffer, 0.5 &#181;L of BSA (1 &#181;g/&#181;L), 1 &#181;L of 10 mM ATP, 1 &#181;L of RNase inhibitor, and 0.5 &#181;L of T4 RNA ligase. The total volume is 4 &#181;L per reaction.</li>
	<li>Add 1 &#181;L of 5&#8217; 20 &#181;M RNA linker to the RNA from step 13.4, denature the RNA by heating at 70 &#176;C for 2 min, and chill on ice immediately.</li>
	<li>Add the 5&#8217; RNA ligation mix to the RNA from step 14.2. Incubate at 16 &#176;C overnight.</li>
</ol>
15. Reverse transcription
<ol>
	<li>Purify the RNAs as described in section 13 and dissolve the RNA with 11.5 &#181;L of RNase-free water.</li>
	<li>Add 1 &#181;L of 10 &#181;M reverse transcription primer to the purified RNA, heat at 70 &#176;C for 2 min, and chill on ice immediately.</li>
	<li>Make a reverse transcription mix with 4 &#181;L of 5x reverse transcription buffer, 1 &#181;L of 10 mM dNTPs, 1 &#181;L of 0.1 M DTT, 1 &#181;L of RNase inhibitor, and 0.5 &#181;L of reverse transcriptase (Table of Materials). The total volume is 7.5 &#181;L per reaction.</li>
	<li>Add 7.5 &#181;L of reverse transcription mix to the RNA, incubate at 50 &#176;C for 30 min, and stop the reaction by incubation at 70 &#176;C for 10 min. Leave at 4 &#176;C.</li>
</ol>
16. PCR amplification
<ol>
	<li>Make a PCR amplification mix with 15 &#181;L of 2x PCR master mix (Table of Materials), 5 &#181;L of cDNA from step 15.4, 0.6 &#181;L of 10 &#181;M forward primer 1 (Table 2), 0.6 &#181;L of 10 &#181;M reverse primer 1 (Table 2), and 8.8 &#181;L of H2O. The total volume is 30 &#181;L.</li>
	<li>Amplify the cDNA with the following settings: 98 &#176;C 30 s, 98 &#176;C 10 s, 60 &#176;C 30 s, 72 &#176;C 30 s (repeat steps 2&#8722;4 for 15&#8722;20 cycles), 72 &#176;C 2 min, and hold at 4 &#176;C. Run the PCR product on a 2&#8722;3% agarose gel. Cut out the DNA of ~100&#8722;150 bp, extract DNA with gel extraction kit (Table of Materials), and elute the DNA with 20 &#181;L of water.</li>
	<li>Take 3 &#181;L of the purified PCR product, amplify with forward primer 2 (Table 2) and reverse primer 2 (index primer; Table 2) as described in step 16.2 for five cycles to introduce index sequences. Purify the PCR product as described in step 16.2.</li>
	<li>Sequence the library with a high-throughput sequencing platform.</li>
</ol>
17. Bioinformatics analysis
<ol>
	<li>Perform data analysis using commercial programs as previously described8.</li>
</ol>

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The authors have nothing to disclose.

Here we introduce a modified CLIP-seq method called FbioCLIP-seq, taking advantage of the FLAG-biotin double tagging system to perform tandem purification of protein-RNA complexes. The FLAG-biotin double tagging system has been shown to be powerful in identifying protein-protein and protein-DNA interactions13,21. Here we demonstrate the high specificity and convenience of this system in identifying the RNAs interacting with proteins. Through tandem pur...

RNAs and RNA-binding proteins (RBPs) control diverse cellular processes including splicing, translation, ribosome biogenesis, epigenetic regulation, and cell fate transition1,2,3,4,5,6. The delicate mechanisms of these processes depend on the unique spatial and temporal arrangement of RNAs and RBPs. Therefore, an important step towards understanding RNA regulation at the molecular level is to reveal the positional information about the binding sites of RBPs.
A strategy referred to as cross-linking and immunoprecipitation (CLIP-seq) has been developed to capture protein-RNA interactions with UV cross-linking followed by immunoprecipitation of the protein of interest7. The key feature of the methodology is the induction of covalent cross-links between an RNA-binding protein and its directly bound RNA molecules (within ~1 &#197;) by UV irradiation8. The RBP footprints can be determined by CLIP tag clustering and peak calling, which usually have a resolution of 30&#8722;60 nt. Alternatively, the reverse transcription step of CLIP can lead to indels (insertions or deletions) or substitutions to the cross-linking sites, which allows identification of protein binding sites on the RNAs at a single-nucleotide resolution. Pipelines like Novoalign and CIMS have been developed for the analysis of the high-throughput sequencing results of CLIP-seq8. Several modified CLIP-seq methods have also been proposed, including individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP), enhanced CLIP (eCLIP), irCLIP, and photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP)9,10,11,12.
Despite the wide use of traditional CLIP-seq methods in the study of RBPs, the CLIP methods have several drawbacks. First, the tedious denatured gel electrophoresis and membrane transfer procedure may lead to loss of information, and cause limited sequence complexity. Second, the protein specific antibody-based CLIP method may pull down a protein complex instead of a single target protein, which may lead to false positive protein-RNA interactions from the copurified RBPs. Third, the antibody-based strategy requires a large amount of high-quality antibodies, which makes the application of these methods inadequate for the study of RBPs without high-quality antibodies available. Fourth, the traditional CLIP method requires radiolabeled ATP to label the protein-bound RNAs.
The high affinity of streptavidin to biotinylated proteins makes it a very powerful approach to purify specific proteins or protein complexes. The efficient biotinylation of proteins carrying an artificial peptide sequence by ectopically expressed bacterial BirA biotin ligase in mammalian cells makes it an efficient strategy to perform biotin purification in vivo13. We developed a modified CLIP-seq method called FbioCLIP-seq (FLAG-Biotin-mediated Cross-linking and Immunoprecipitation followed by high-throughput sequencing) using FLAG-biotin tag tandem purification14 (Figure 1). Through tandem purification and stringent wash conditions, almost all the interacting RBPs are removed (Figure 2). The stringent wash conditions also allow circumventing the SDS-PAGE and membrane transfer, which is labor intensive and technically challenging. And similar to eCLIP and irCLIP, the FbioCLIP-seq method is isotope-free. Skipping the gel running and transfer steps avoids the loss of information, keeps authentic protein-RNA interactions intact, and increases the library complexity. Moreover, the high efficiency of the tagging system makes it a good choice for RBPs without high-quality antibodies available.
Here we provide a step-by-step description of the FbioCLIP-seq protocol for mammalian cells. Briefly, cells are cross-linked by 254 nm UV, followed by cell lysis and FLAG immunoprecipitation (FLAG-IP). Next, the protein-RNA complexes are further purified by biotin affinity capture and RNAs are fragmented by partial digestion with MNase. Then, the protein-bound RNA is dephosphorylated and ligated with a 3&#8217; linker. A 5&#8217; RNA linker is added after the RNA is phosphorylated with PNK and eluted by proteinase K digestion. After reverse transcription, the protein-bound RNA signals are amplified by PCR and purified by agarose gel purification. Two RBPs were chosen to exemplify the FbioCLIP-seq result. LIN28 is a well-characterized RNA-binding protein involved in microRNA maturation, protein translation, and cell reprogramming15,16,17. WDR43 is a WD40 domain-containing protein thought to coordinate ribosome biogenesis, eukaryotic transcription, and embryonic stem cell pluripotency control14,18. Consistent with previously reported results for LIN28 with CLIP-seq, FbioCLIP-seq reveals binding sites of LIN28 on &#8220;GGAG&#8221; motifs in the microRNA mir-let7g and mRNAs16,19 (Figure 3). WDR43 FbioCLIP-seq also identified the binding preference of WDR43 with 5&#39; external transcribed spacers (5&#39;-ETS) of pre-rRNAs20 (Figure 4). These results validate the reliability of the FbioCLIP-seq method.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>UV crosslinker</td><td>UVP</td><td>HL-2000 HybrilLinker</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Affinity Purification Beads&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ANTI-FLAG beads</td><td>Sigma-Aldrich</td><td>A2220</td><td></td></tr><tr><td>Streptavidin beads</td><td>Invitrogen</td><td>112.06D</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10x PBS</td><td>Gibco</td><td>70013032</td><td></td></tr><tr><td>3 M NaOAc</td><td>Ambion</td><td>AM9740</td><td></td></tr><tr><td>3 x FLAG peptide</td><td>Sigma-Aldrich</td><td>F4799</td><td></td></tr><tr><td>ATP</td><td>Sigma-Aldrich</td><td>A6559</td><td></td></tr><tr><td>Calcium chloride (CaCl2)</td><td>Sigma-Aldrich</td><td>C1016</td><td></td></tr><tr><td>CIP</td><td>NEB</td><td>M0290S</td><td>CIP buffer is in the same package.</td></tr><tr><td>DTT</td><td>Sigma-Aldrich</td><td>D0632</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E9884</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E3889</td><td></td></tr><tr><td>Gel purification kit</td><td>QIAGEN</td><td>28704</td><td></td></tr><tr><td>Glycogen</td><td>Ambion</td><td>AM9510</td><td></td></tr><tr><td>Magnesium chloride (MgCl2)</td><td>Sigma-Aldrich</td><td>449172</td><td></td></tr><tr><td>MNase</td><td>NEB</td><td>M0247S</td><td></td></tr><tr><td>NP-40</td><td>Amresco</td><td>M158-500ML</td><td></td></tr><tr><td>PMSF</td><td>Sigma-Aldrich</td><td>10837091001</td><td></td></tr><tr><td>Porteinase K</td><td>TAKARA</td><td>9033</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>P8340</td><td></td></tr><tr><td>Q5 High-Fidelity 2X Master Mix</td><td>NEB</td><td>0492S</td><td></td></tr><tr><td>reverse trancriptase (SupperScriptIII)</td><td>Invitrogen</td><td>18080093</td><td></td></tr><tr><td>RNA isolation reagent (Trizol)</td><td>Invitrogen</td><td>15596018</td><td></td></tr><tr><td>RNase Inhibitor</td><td>ThermoFisher</td><td>EO0381</td><td></td></tr><tr><td>RNaseOUT</td><td>Invitrogen</td><td>10777019</td><td></td></tr><tr><td>RQ1 Dnase</td><td>Promega</td><td>M6101</td><td></td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>1614363</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich</td><td>D6750</td><td></td></tr><tr><td>T4 PNK</td><td>NEB</td><td>M0201S</td><td>PNK buffer is in the same package.</td></tr><tr><td>T4 RNA ligaes</td><td>ThermoFisher</td><td>EL0021</td><td>T4 RNA ligase buffer and BSA are in the same package.</td></tr><tr><td>T4 RNA ligase2, truncated</td><td>NEB</td><td>M0242S</td><td>T4 RNA ligase buffer and 50% PEG are in the same package.</td></tr><tr><td>Trypsin-EDTA</td><td>ThermoFisher</td><td>25200072</td><td></td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>208884</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;mESC culture medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM (80%)</td><td>Gibco</td><td>11965126</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Gibco</td><td>21985023</td><td></td></tr><tr><td>FCS (15%)</td><td>Hyclone</td><td></td><td></td></tr><tr><td>Glutamax (1%)</td><td>Gibco</td><td>35050061</td><td></td></tr><tr><td>LIF</td><td></td><td></td><td>purified recombinant protein; 10,000 fold dilution</td></tr><tr><td>NEAA (1%)</td><td>Gibco</td><td>11140050</td><td></td></tr><tr><td>Nucleoside mix (1%)</td><td>Millipore</td><td>ES-008-D</td><td></td></tr><tr><td>Penicillin-Streptomycin (1%)</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Kit&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DNA gel extraction kit</td><td>QIAGEN</td><td>28704</td><td></td></tr></tbody></table>

transcriptome-wide profiling of protein-rna interactions by cross-linking and immunoprecipitation mediated by flag-biotin tandem purification

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate regulation of these processes. A strategy called CLIP-seq (cross-linking and immunoprecipitation) has been developed to capture endogenous protein-RNA interactions with UV cross-linking followed by immunoprecipitation. Despite the wide use of conventional CLIP-seq method in RBP study, the CLIP method is limited by the availability of high-quality antibodies, potential contaminants from the copurified RBPs, requirement of isotope manipulation, and potential loss of information during a tedious experimental procedure. Here we describe a modified CLIP-seq method called FbioCLIP-seq using the FLAG-biotin tag tandem purification. Through tandem purification and stringent wash conditions, almost all the interacting RNA-binding proteins are removed. Thus, the RNAs interacting indirectly mediated by these copurified RBPs are also reduced. Our FbioCLIP-seq method allows efficient detection of direct protein-bound RNAs without SDS-PAGE and membrane transfer procedures in an isotope-free and protein-specific antibody-free manner.

The schematic representation of the FbioCLIP-seq procedure is shown in Figure 1. Compared with FLAG-mediated or streptavidin-mediated one-step affinity purification, FLAG-biotin tandem purification removed almost all the copurified proteins, avoiding the contamination of indirect protein-RNA interactions (Figure 2). Representative results for FbioCLIP-seq for LIN28 and WDR43 are depicted in Figure 3 and <strong...

Watch this Scientific Journal Video about Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification at JoVE.com

Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification

Here we present a modified CLIP-seq protocol called FbioCLIP-seq with FLAG-biotin tandem purification to determine the RNA targets of RNA-binding proteins (RBPs) in mammalian cells.

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate ...

transcriptome-wide-profiling-protein-rna-interactions-cross-linking

Research

JoVE Journal

Genetics

5.8K Views.  Tsinghua University. Here we present a modified CLIP-seq protocol called FbioCLIP-seq with FLAG-biotin tandem purification to determine the RNA targets of RNA-binding proteins (RBPs) in mammalian cells.

Video: Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Biochemistry

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3&#39;-biotinylated 2&#39;-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.

An easy-to-use RNA pull-down protocol is designed for the identification of RNAs engaged in direct RNA/RNA interaction with a long non-coding RNA. The protocol uses psoralen as a fixative to cross-link only RNA/RNA interactions and provides the whole direct RNA interactome of a long non-coding RNA when coupled with RNA sequencing.

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome ...

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be bound by many different RBPs that will regulate its production, stability, activity, and degradation. As such, much has been done to understand the dynamics that exist between these two types of molecules. A particularly important breakthrough came with the emergence of &#8216;cross-linking and immunoprecipitation&#8217; (CLIP). This technique allowed stringent investigation into which RNAs are bound by a particular RBP. In short, the protein of interest is UV cross-linked to its RNA substrates in vivo, purified under highly stringent conditions, and then the RNAs covalently cross-linked to the protein are converted into cDNA libraries and sequenced. Since its conception, many derivative techniques have been developed in order to make CLIP amenable to particular fields of study. However, cross-linking using ultraviolet light is notoriously inefficient. This results in extended exposure times that make the temporal study of RBP-RNA interactions impossible. To overcome this issue, we recently designed and built much-improved UV irradiation and cell harvesting devices. Using these new tools, we developed a protocol for time-resolved analyses of RBP-RNA interactions in living cells at high temporal resolution: Kinetic CRoss-linking and Analysis of cDNAs (&#967;CRAC). We recently used this technique to study the role of yeast RBPs in nutrient stress adaptation. This manuscript provides a detailed overview of the &#967;CRAC method and presents recent results obtained with the Nrd1 RBP.

Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Biology

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying the binding partners of an RNA of interest remains of high importance to unveil the mechanisms behind many cellular processes. However, RNA molecules might interact transiently and dynamically with some RNA-binding proteins (RBPs), especially with non-canonical ones. Hence, improved methods to isolate and identify such RBPs are greatly needed.
To identify the protein partners of a known RNA sequence efficiently and quantitatively, we developed a method based on the pull-down and characterization of all interacting proteins, starting from cellular total protein extract. We optimized the protein pull-down using biotinylated RNA pre-loaded on streptavidin-coated beads. As a proof of concept, we employed a short RNA sequence known to bind the neurodegeneration-associated protein TDP-43 and a negative control of a different nucleotide composition but the same length. After blocking the beads with yeast tRNA, we loaded the biotinylated RNA sequences on the streptavidin beads and incubated them with the total protein extract from HEK 293T cells. After incubation and several washing steps to remove nonspecific binders, we eluted the interacting proteins with a high-salt solution, compatible with the most commonly used protein quantification reagents and with sample preparation for mass spectrometry. We quantified the enrichment of TDP-43 in the pull-down performed with the known RNA binder compared to the negative control by mass spectrometry. We used the same technique to verify the selective interactions of other proteins computationally predicted to be unique binders of our RNA of interest or of the control. Finally, we validated the protocol by western blot via the detection of TDP-43 with an appropriate antibody. 
This protocol will allow the study of the protein partners of an RNA of interest in near-to-physiological conditions, helping uncover unique and unpredicted protein-RNA interactions.

Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis.

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.