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>Equipment</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>Affinity Purification Beads</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>Reagents</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>mESC culture medium</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>Kit</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

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in production or degradation of RNA. This protocol describes a method for measurement of RNA production, using 5-Bromouridine labelling of RNA followed by immunoprecipitation, which enables investigation of RNA synthesized within a short timeframe (e.g., 1 h). The advantage of 5-Bromouridine-labelling and immunoprecipitation over the use of toxic transcriptional inhibitors, such as &#945;-amanitin and actinomycin D, is that there are no or very low effects on cell viability during short-term use. However, because 5-Bromouridine-immunoprecipitation only captures RNA produced within the short labelling time, slowly produced as well as rapidly degraded RNA can be difficult to measure by this method. The 5-Bromouridine-labelled RNA captured by 5-Bromouridine-immunoprecipitation can be analyzed by reverse transcription, quantitative polymerase chain reaction, and next generation sequencing. All types of RNA can be investigated, and the method is not limited to measuring mRNA as is presented in this example.

This method can be used to measure RNA synthesis. 5-Bromouridine is added to cells and incorporated into synthesized RNA. RNA synthesis is measured by RNA extraction immediately after labelling, followed by 5-Bromouridine-targeted immunoprecipitation of labelled RNA and analysis by reverse transcription and quantitative polymerase chain reaction.

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of a wide range of transcription factors (TFs) and cofactors mediating transcriptional activation or repression via changes in chromatin structure. Chromatin immunoprecipitation (ChIP) is a useful molecular biology approach for mapping histone modifications and profiling transcription factors/cofactors binding to DNA, thus providing a snapshot of the dynamic nuclear changes occurring during different biological processes.
To study transcriptional regulation in adipose tissue, samples derived from in vitro cell cultures of immortalized or primary cell lines are often favored in ChIP assays because of the abundance of starting material and reduced biological variability. However, these models represent a limited snapshot of the actual chromatin state in living organisms. Thus, there is a critical need for optimized protocols to perform ChIP on adipose tissue samples derived from animal models.
Here we describe a protocol for efficient ChIP-seq of both histone modifications and non-histone proteins in brown adipose tissue (BAT) isolated from a mouse. The protocol is optimized for investigating genome-wide localization of proteins of interest and epigenetic markers in the BAT, which is a morphologically and physiologically distinct tissue amongst fat depots.

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo.

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of ...

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

Yersinia ruckeri is an important pathogen of farmed salmonids worldwide, but simple tools suitable for epizootiological investigations (infection tracing, etc.) of this bacterium have been lacking. A Multi-Locus Variable-number tandem-repeat Analysis (MLVA) assay was therefore developed as an easily accessible and unambiguous tool for high-resolution genotyping of recovered isolates. For the MLVA assay presented here, DNA is extracted from cultured Y. ruckeri samples by boiling bacterial cells in water, followed by use of supernatant as template for PCR. Primer-pairs targeting ten Variable-number tandem-repeat (VNTR) loci, interspersed throughout the Y. ruckeri genome, are distributed equally amongst two five-plex PCR reactions running under identical cycling conditions. Forward primers are labelled with either of three fluorescent dyes. Following amplicon confirmation by gel electrophoresis, PCR products are diluted and subjected to capillary electrophoresis. From the resulting electropherogram profiles, peaks representing each of the VNTR loci are size-called and employed for calculating VNTR repeat counts in silico. Resulting ten-digit MLVA profiles are then&#160;used to generate Minimum spanning trees enabling epizootiological evaluation by cluster analysis. The highly portable output data, in the form of numerical MLVA profiles, can rapidly be compared across labs and placed in a spatiotemporal context. The entire procedure from cultured colony to epizootiological evaluation may be completed for up to 48 Y. ruckeri isolates within a single working day.

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

The Multi-Locus Variable-number tandem-repeat Analysis (MLVA) assay presented here enables inexpensive, robust and portable high-resolution genotyping of the fish-pathogenic bacterium Yersinia ruckeri. Starting from pure cultures, the assay employs multiplex PCR and capillary electrophoresis to produce ten-loci MLVA profiles for downstream applications.

Yersinia ruckeri is an important pathogen of farmed salmonids worldwide, but simple tools suitable for epizootiological investigations (infection tracing, ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

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 ...

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the only known non-legume lineage able to establish a nitrogen-fixing nodule symbiosis with rhizobium. Comparative studies between legumes and P. andersonii could provide valuable insight into the genetic networks underlying root nodule formation. To facilitate comparative studies, we recently sequenced the P. andersonii genome and established Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing. Here, we provide a detailed description of the transformation and genome editing procedures developed for P. andersonii. In addition, we describe procedures for the seed germination and characterization of symbiotic phenotypes. Using this protocol, stable transgenic mutant lines can be generated in a period of 2-3 months. Vegetative in vitro propagation of T0 transgenic lines allows phenotyping experiments to be initiated at 4 months after A. tumefaciens co-cultivation. Therefore, this protocol takes only marginally longer than the transient Agrobacterium rhizogenes-based root transformation method available for P. andersonii, though offers several clear advantages. Together, the procedures described here permit P. andersonii to be used as a research model for studies aimed at understanding symbiotic associations as well as potentially other aspects of the biology of this tropical tree.

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae) and can form nitrogen-fixing root nodules in association with the rhizobium. Here, we describe a detailed protocol for reverse genetic analyses in P. andersonii based on Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing.

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the ...

Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat (Fagopyrum tataricum)

Tartary buckwheat (TB) [Fagopyrum tataricum (L.) Gaertn] possesses various biological and pharmacological activities because it contains abundant secondary metabolites such as flavonoids, especially rutin. Agrobacterium rhizogenes have been gradually used worldwide to induce hairy roots in medicinal plants to investigate gene functions and increase the yield of secondary metabolites. In this study, we have described a detailed method to generate A. rhizogenes-mediated hairy roots in TB. Cotyledons and hypocotyledonary axis at 7&#8211;10 days were selected as explants and infected with A. rhizogenes carrying a binary vector, which induced adventitious hairy roots that appeared after 1 week. The generated hairy root transformation was identified based on morphology, resistance selection (kanamycin), and reporter gene expression (green fluorescent protein). Subsequently, the transformed hairy roots were self-propagated as required. Meanwhile, a myeloblastosis (MYB) transcription factor, FtMYB116, was transformed into the TB genome using the A. rhizogenes-mediated hairy roots to verify the role of FtMYB116 in synthesizing flavonoids. The results showed that the expression of flavonoid-related genes and the yield of flavonoid compounds (rutin and quercetin) were significantly (p &#60; 0.01) promoted by FtMYB116, indicating that A. rhizogenes-mediated hairy roots can be used as an effective alternative tool to investigate gene functions and the production of secondary metabolites. The detailed step-by-step protocol described in this study for generating hairy roots can be adopted for any genetic transformation or other medicinal plants after adjustment.

Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat Fagopyrum tataricum

We describe a method of inducing hairy roots by Agrobacterium rhizogenes-mediated transformation in Tartary buckwheat (Fagopyrum tataricum). This can be used to investigate gene functions and production of secondary metabolites in Tartary buckwheat, be adopted for any genetic transformation, or used for other medicinal plants after improvement.