Name: Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification (Video) | JoVE Journal
Uploaded: 2020-05-18T13:00:00.000+00:00
Duration: 8 min 46 s
Description: Learn Fbio CLIP-seq for profiling protein-RNA interactions in mammalian cells using FLAG-biotin tandem purification. This isotope-free method efficiently detects direct protein-bound RNAs without SDS-PAGE.

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5 minutes

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>

<ol><li>Kim, B., Jeong, K., Kim, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+Mapping+of+DROSHA+Cleavage+Sites+on+Primary+MicroRNAs+and+Noncanonical+Substrates%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">Genome-wide Mapping of DROSHA Cleavage Sites on Primary MicroRNAs and Noncanonical Substrates.</a> Molecular Cell. 66 (2), 258-269 (2017).</li>
<li>Guallar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA-dependent+chromatin+targeting+of+TET2+for+endogenous+retrovirus+control+in+pluripotent+stem+cells%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">RNA-dependent chromatin targeting of TET2 for endogenous retrovirus control in pluripotent stem cells.</a> Nature Genetics. 50 (3), 443-451 (2018).</li>
<li>Holmqvist, E., Li, L., Bischler, T., Barquist, L., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+Maps+of+ProQ+Binding+In+Vivo+Reveal+Target+Recognition+via+RNA+Structure+and+Stability+Control+at+mRNA+3%27+Ends%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">Global Maps of ProQ Binding In Vivo Reveal Target Recognition via RNA Structure and Stability Control at mRNA 3' Ends.</a> Molecular Cell. 70 (5), 971-982 (2018).</li>
<li>Wei, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RBFox2+Binds+Nascent+RNA+to+Globally+Regulate+Polycomb+Complex+2+Targeting+in+Mammalian+Genomes%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">RBFox2 Binds Nascent RNA to Globally Regulate Polycomb Complex 2 Targeting in Mammalian Genomes.</a> Molecular Cell. 62 (6), 875-889 (2016).</li>
<li>Kim, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+PARP-1+by+snoRNAs+Controls+Ribosome+Biogenesis+and+Cell+Growth+via+the+RNA+Helicase+DDX21%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">Activation of PARP-1 by snoRNAs Controls Ribosome Biogenesis and Cell Growth via the RNA Helicase DDX21.</a> Molecular Cell. 75 (6), 1270-1285 (2019).</li>
<li>Yao, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nascent+Pre-rRNA+Sorting+via+Phase+Separation+Drives+the+Assembly+of+Dense+Fibrillar+Components+in+the+Human+Nucleolus%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">Nascent Pre-rRNA Sorting via Phase Separation Drives the Assembly of Dense Fibrillar Components in the Human Nucleolus.</a> Molecular Cell. 76 (5), 767-783 (2019).</li>
<li>Licatalosi, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HITS-CLIP+yields+genome-wide+insights+into+brain+alternative+RNA+processing%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">HITS-CLIP yields genome-wide insights into brain alternative RNA processing.</a> Nature. 456 (7221), 464-469 (2008).</li>
<li>Moore, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+Argonaute+and+conventional+RNA-binding+protein+interactions+with+RNA+at+single-nucleotide+resolution+using+HITS-CLIP+and+CIMS+analysis%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis.</a> Nature Protocols. 9 (2), 263-293 (2014).</li>
<li>Konig, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((iCLIP--transcriptome-wide+mapping+of+protein-RNA+interactions+with+individual+nucleotide+resolution%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">iCLIP--transcriptome-wide mapping of protein-RNA interactions with individual nucleotide resolution.</a> Journal of Visualized Experiments. (50), e2638(2011).</li>
<li>Zarnegar, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((irCLIP+platform+for+efficient+characterization+of+protein-RNA+interactions%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">irCLIP platform for efficient characterization of protein-RNA interactions.</a> Nature Methods. 13 (6), 489-492 (2016).</li>
<li>Van Nostrand, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Robust+transcriptome-wide+discovery+of+RNA-binding+protein+binding+sites+with+enhanced+CLIP+%28eCLIP%29%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP).</a> Nature Methods. 13 (6), 508-514 (2016).</li>
<li>Hafner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PAR-CliP--a+method+to+identify+transcriptome-wide+the+binding+sites+of+RNA+binding+proteins%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">PAR-CliP--a method to identify transcriptome-wide the binding sites of RNA binding proteins.</a> Journal of Visualized Experiments. (41), e2034(2010).</li>
<li>de Boer, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+biotinylation+and+single-step+purification+of+tagged+transcription+factors+in+mammalian+cells+and+transgenic+mice%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (13), 7480-7485 (2003).</li>
<li>Bi, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA+Targets+Ribogenesis+Factor+WDR43+to+Chromatin+for+Transcription+and+Pluripotency+Control%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">RNA Targets Ribogenesis Factor WDR43 to Chromatin for Transcription and Pluripotency Control.</a> Molecular Cell. 75 (1), 102-116 (2019).</li>
<li>Heo, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TUT4+in+concert+with+Lin28+suppresses+microRNA+biogenesis+through+pre-microRNA+uridylation%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation.</a> Cell. 138 (4), 696-708 (2009).</li>
<li>Cho, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LIN28A+Is+a+Suppressor+of+ER-Associated+Translation+in+Embryonic+Stem+Cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">LIN28A Is a Suppressor of ER-Associated Translation in Embryonic Stem Cells.</a> Cell. 151 (4), 765-777 (2012).</li>
<li>Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318 (5858), 1917-1920 (2007).</li>
<li>Zhao, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+specific+roles+for+the+ribosome+biogenesis+factor+Wdr43+in+zebrafish+development%5BTitle%5D)+AND+%22PLoS+Genetics%22%5BJournal%5D)">Tissue specific roles for the ribosome biogenesis factor Wdr43 in zebrafish development.</a> PLoS Genetics. 10 (1), 1004074(2014).</li>
<li>Wilbert, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LIN28+binds+messenger+RNAs+at+GGAGA+motifs+and+regulates+splicing+factor+abundance%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">LIN28 binds messenger RNAs at GGAGA motifs and regulates splicing factor abundance.</a> Molecular Cell. 48 (2), 195-206 (2012).</li>
<li>Hunziker, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((UtpA+and+UtpB+chaperone+nascent+pre-ribosomal+RNA+and+U3+snoRNA+to+initiate+eukaryotic+ribosome+assembly%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">UtpA and UtpB chaperone nascent pre-ribosomal RNA and U3 snoRNA to initiate eukaryotic ribosome assembly.</a> Nature Communications. 7, 12090(2016).</li>
<li>Kim, J., Cantor, A. B., Orkin, S. H., Wang, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+in+vivo+biotinylation+to+study+protein-protein+and+protein-DNA+interactions+in+mouse+embryonic+stem+cells%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Use of in vivo biotinylation to study protein-protein and protein-DNA interactions in mouse embryonic stem cells.</a> Nature Protocols. 4 (4), 506-517 (2009).</li>
<li>Li, Z., Michael, I. P., Zhou, D., Nagy, A., Rini, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simple+piggyBac+transposon-based+mammalian+cell+expression+system+for+inducible+protein+production%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Simple piggyBac transposon-based mammalian cell expression system for inducible protein production.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (13), 5004-5009 (2013).</li>
<li>Hnasko, T. S., Hnasko, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Western+Blot%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">The Western Blot.</a> Methods in Molecular Biology. 1318, 87-96 (2015).</li>
<li>Aktas, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DHX9+suppresses+RNA+processing+defects+originating+from+the+Alu+invasion+of+the+human+genome%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">DHX9 suppresses RNA processing defects originating from the Alu invasion of the human genome.</a> Nature. 544 (7648), 115-119 (2017).</li>
<li>Xu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+Crosslinking+Immunoprecipitation+%28eCLIP%29+Method+for+Efficient+Identification+of+Protein-bound+RNA+in+Mouse+Testis%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Enhanced Crosslinking Immunoprecipitation (eCLIP) Method for Efficient Identification of Protein-bound RNA in Mouse Testis.</a> Journal of Visualized Experiments. (147), e59681(2019).</li>
</ol>

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

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...

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

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.

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

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.

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

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

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.

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

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.

mRNA Interactome Capture from Plant Protoplasts

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.

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

Here, we present a protocol to enrich endogenous RNA binding sites or "footprints" 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).

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

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.

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

Co-translational interactions play a crucial role in nascent-chain modifications, targeting, folding, and assembly pathways. Here, we describe Selective Ribosome Profiling, a method for in vivo, direct analysis of these interactions in the model eukaryote Saccharomyces cerevisiae.

Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling

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.

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

Learn Fbio CLIP-seq for profiling protein-RNA interactions in mammalian cells using FLAG-biotin tandem purification. This isotope-free method efficiently detects direct protein-bound RNAs without SDS-PAGE.

Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification (Video) | JoVE Journal

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

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