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

1, 2, 3, 3, 4, 4, 1, 1, 2, 2, 1, 1, 5, 3, 1

1Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, Rockefeller University, 2Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, 3Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), 4Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), 5Genomics Resource Center, Rockefeller University

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    Summary

    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.

    Date Published: 7/02/2010, Issue 41; doi: 10.3791/2034

    Cite this Article

    Hafner, M., Landthaler, M., Burger, L., Khorshid, M., Hausser, J., Berninger, P., et al. PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins. J. Vis. Exp. (41), e2034, doi:10.3791/2034 (2010).

    Abstract

    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

    Protocol

    The protocol below describes the PAR-CliP procedure for HEK293 cells expressing FLAG/HA-tagged IGF2BP1 upon induction with doxycycline. We will use an anti-FLAG antibody for immunoprecipitation.

    PAR-CliP will work with any cell line expressing detectable levels of the endogenous, untagged RNA binding protein (RBP) of interest if an efficient antibody for immunoprecipitation is available.

    Expanding Cells

    1. Expand FlpIn-HEK293/TO/FLAG/HA-IGF2BP1 cells in growth medium. We recommend using between 100-400 x 106 cells (approx. 10-40 15 cm cell culture plates) as a starting point. Grow them to approximately 80% confluency.
    2. 14 h before crosslinking add a) 4-thiouridine to a final concentration of 100 μM (1:1000 v/v of a 1 M 4-thiouridine stock solution) directly to the cell culture medium and b) induce expression of the FLAG/HA tagged IGF2BP1 by addition of 1 μg/ml of doxycycline (1:10,000 v/v of 10 mg/ml doxycycline stock solution). NOTE: instead of 4-thiouridine you can also use 100 μM of 6-thioguanosine.

    UV-Crosslinking

    1. Wash cells once with 10 ml ice-cold PBS per plate and remove PBS completely.
    2. Place plates on a tray with ice and irradiate uncovered with 0.15 J/cm2 of 365 nm UV light in a Stratalinker 2400 (Stratagene) or similar device.
    3. Scrape cells off with a rubber policeman in 1 ml PBS per plate, transfer to 50 ml centrifugation tubes and collect by centrifugation at 500 x g for 5 min at 4°C and discard the supernatant 100 x 106 HEK293 cells (10 15 cm plates) will yield approx. 1 ml of wet cell pellet.
    4. (optional) Unless you want to continue directly with cell lysis, shock freeze the cell pellet in liquid nitrogen and store at -80°C Cell pellets can be stored for at least 12 months.

    Cell lysis and RNaseT1 digest

    1. Take up cell pellet of crosslinked cells in 3 volumes of 1x NP40 lysis buffer and incubate on ice for 10 min.
    2. Clear cell lysate by centrifugation at 13,000 x g for 15 min at 4°C.
    3. Clear the lysate further by filtering it through a 0.2 μm membrane syringe filter (Pall Acrodisc or equivalent).
    4. Add RNase T1 (Fermentas, 10,000 U/μl) to a final concentration of 1 U/μl and incubate in a water bath for 15 min at 22°C. Cool reaction subsequently for 5 min on ice before proceeding.

    Immunoprecipitation and recovery of crosslinked target RNA fragments

    Using the magnetic separator

    Follow these guidelines throughout the sample preparation to prevent the magnetic beads from drying out.

    1. Place the tube containing the beads on the magnetic stand for 1 2 minutes.
    2. Add the buffer to the tube while the tube is on the magnetic separator.
    3. Cap the tube, remove it from the magnetic separator, and resuspend the beads. You can resuspend the beads by flicking the tube with your finger or use a vortexer set at 5 6.
    4. Centrifuge briefly to collect any beads that may remain in the tube cap.
    5. Repeat steps 1 through 4 as required.

    Preparation of magnetic beads

    1. Transfer 10 μl of Dynabeads Protein G magnetic particles (Invitrogen) per ml cell lysate (for a typical experiment it should be approx. 40 50 μl of beads) to a 1.5 ml microfuge tube. Wash beads twice with 1 ml of citrate-phosphate buffer.
    2. Resuspend in twice the volume of citrate-phosphate buffer relative to the original volume of bead suspension.
    3. Add 0.25 μg of anti-FLAG M2 monoclonal antibody (Sigma) per ml suspension and incubate on a rotating wheel for 40 min at room temperature.
    4. Wash beads twice in 1 ml of citrate-phosphate buffer to remove unbound antibody.
    5. Resuspend beads in twice the volume of citrate-phosphate buffer relative to the original volume of bead suspension.

    Immunoprecipitation (IP), second RNase T1 digestion, and dephosphorylation

    1. Add 20 μl of freshly prepared antibody-conjugated magnetic beads per ml of partial RNase T1 treated cell lysate and incubate in 15 ml centrifugation tubes on a rotating wheel for 1 h at 4°C.
    2. Collect magnetic beads on a magnetic particle collector for 15 and 50 ml centrifugation tubes (Invitrogen) and transfer to 1.5 ml microfuge tubes.
    3. Wash beads 3 times in 1 ml of IP wash buffer.
    4. Add RNaseT1 (Fermentas, 10,000 U/μl) to a final concentration of 100 U/μl and incubate the bead suspension in a water bath for 15 min at 22 °C. Cool subsequently on ice for 5 min.
    5. Wash beads 3 times in 1 ml of high-salt wash buffer.
    6. Resuspend beads in 1 volume of dephosphorylation buffer
    7. Add calf intestinal alkaline phosphatase (CIAP, NEB) to a final concentration of 0.5 U/μl, and incubate the suspension for 10 min at 37°C.
    8. Wash beads twice in 1 ml of phosphatase wash buffer
    9. Wash beads twice in polynucleotide kinase (PNK) buffer without DTT (the DTT concentration necessary for the enzymatic reaction is high enough to damage the magnetic beads).
    10. Resuspend beads in one original bead volume of PNK buffer

    Radiolabeling of RNA segments crosslinked to immunoprecipitated proteins

    1. To the bead suspension described above, add Υ-32P-ATP to a final concentration of 0.5 μCi/μl and T4 PNK (NEB) to 1 U/μl in one original bead volume. Incubate the suspension for 30 min at 37°C.
    2. Add non-radioactive ATP to obtain a final concentration of 100 μM and incubate for another 5 min at 37°C.
    3. Wash the magnetic beads 5 times with 800 μl of PNK buffer without DTT.
    4. Resuspend the beads in 70 μl of SDS-PAGE loading buffer.

    SDS-PAGE and electroelution of crosslinked RNA-protein complexes from gel slices

    1. Incubate the radiolabeled suspension for 5 min in a heat block at 95°C to denature and release the immunoprecipitated RBP with crosslinked RNA and vortex.
    2. Remove the magnetic beads on the separator and transfer the supernatant to a clean 1.5 ml microfuge tube.
    3. Load 40 μl of the supernatant per well of a Novex Bis-Tris 4-12% (Invitrogen) precast polyacrylamide gel and run the gel for 55 min at 200 V.
    4. Disassemble the gel chamber and dismantle the gel, leaving it mounted on one plate. To facilitate the alignment of the gel to the phosphorimager paper printout, we recommend implanting three tiny radioactive gel pieces asymmetrically at three of the four corners of the gel. Radioactive gel pieces can be collected from gels that were previously used to purify radiolabeled synthetic oligonucleotides Wrap the gel in plastic film (e.g. Saran wrap) to avoid contamination.
    5. Expose the gel to a blanked phosphorimager screen for 1 h and visualize on a phosphorimager.
    6. Align the gel on top of the phosphorimager printout using the implanted gel pieces for orientation. Cut out the bands that correspond to the expected size of RBP (IGF2BP1, approx. 75 kDa) and transfer to a D-Tube Dialyzer Midi Tube and add 800 μl 1x SDS running buffer.
    7. Electroelute the crosslinked RNA-RBP complex in 1x SDS running buffer at 100 V for 2 h. excised from the gel and electroeluted in a D-Tube Dialyzer Midi (Novagen) in 800 μl SDS running buffer according to the instructions of the manufacturer.

    Proteinase K digestion

    1. Add an equal volume of 2x Proteinase K Buffer with respect to the electroeluate, followed by the addition of Proteinase K (Roche) to a final concentration of 1.2 mg/ml. Incubate for 30 min at 55°C.
    2. Recover the RNA by acidic phenol/chloroform/IAA extraction (25:24:1, pH 4.0) followed by a chloroform extraction. Add 1 μl of glycogen (10 mg/ml stock) precipitate the RNA by adding 3 volumes of ethanol. Dissolve the pellet in 10.5 μl water.

    cDNA library preparation and deep sequencing

    Carry the recovered RNA through a standard cDNA library preparation protocol originally described for cloning of small regulatory RNAs 19. The first step, 3' adapter ligation, was carried out as described on a 20 μl scale using 10.5 μl of the recovered RNA. Use the Solexa sequencing adapter sets described. Depending on the amount of RNA recovered, 5'-adapter-3'-adapter products without inserts may be detected after amplification of the cDNA as additional PCR bands. In such cases, excise the longer PCR product of expected size from a 3% NuSieve low-melting point agarose gel, elute the PCR product from the gel pieces using the GelElute kit (Qiagen) and sequence using the Solexa technology. One Solexa sequencing run usually affords between 6 and 10 million sequence reads that are enough for a transcriptome wide coverage of the binding sites of RNA binding proteins.

    Bioinformatic analysis

    Careful bioinformatic analysis of the sequence reads needs to be done to obtain meaningful insights into the RNA binding sites for the examined RBP, such as the RNA recognition element, the preferred binding regions the RBP has (exonic vs. intronic, coding sequence vs. untranslated sequence). The sequence reads need to be aligned against the genome and EST databases. We usually use reads mapping uniquely to the genome with up to one mismatch, insertion or deletion to build clusters of sequence reads that can then be further analyzed. The frequency of characteristic mutations in the clustered sequenced reads, T to C transitions when using 4-SU and G to A transitions when using 6-SG, are indicative of successfully crosslinked sequences. In our experience uncrosslinked RNAs labeled with 4-SU show a background mutation rate of approximately 20%. This rate is increases to approx. 50-80% upon crosslinking.

    A detailed description of the bioinformatic analysis can be found in the Supplementary material of the publication by Hafner et al.18

    Optional Steps

    Determination of incorporation levels of 4-thiouridine into total RNA

    Isolate total RNA from the cell line stably expressing the RBP of interest after growing in medium supplemented with 100 μM 4SU 16 h prior to harvest. As a control, harvest cells grown without 4SU addition. Isolate total RNA by addition of 3 volumes of Trizol reagent (Sigma) to the washed cell pellets following the manufacturer s instructions. was Further purify total RNA using Qiagen RNeasy according to the manufacturer's protocol. To prevent oxidization of 4SU during RNA isolation and analysis, add 0.1 mM dithiothreitol (DTT) to the wash buffers and subsequent enzymatic steps. Digest and dephosphorylated total RNA to single nucleosides for HPLC analysis as described before 20. Briefly, in a 30 μl volume, incubate 40 μg of purified total RNA were for 16 h at 37°C with 0.4 U bacterial alkaline phosphatase (Worthington Biochemical) and 0.09 U snake venom phosphodiesterase (Worthington Biochemical). As a reference standard, use a synthetic 4SU-labeled RNA, (we standardly use CGUACGCGGAAUACUUCGA(4SU)U) and also subject it to complete enzymatic digestion. Separate the resulting mixtures of ribonucleosides by HPLC on a Supelco Discovery C18 (bonded phase silica 5 μM particle, 250 x 4.6 mm) reverse phase column (Bellefonte PA, USA). HPLC buffers are 0.1 M TEAA in 3% acetonitrile (A) and 90% acetonitrile in water (B). Use an isocratic gradient: 0% B for 15 min, 0 to 10 % B for 20 min, 10 to 100% B for 30 min. Apply a 5 min 100 % B wash applied between runs to clean the HPLC column.

    Representative Results

    Figure 1
    Figure 1 (right panel) shows a representative result of a PAR-CliP performed with cell lines expressing FLAG/HA-tagged IGF2BP1 with 4-SU and 6-SG. Note that the crosslinking efficiency of 6-SG for IGF2BP1 is lower than the crosslinking efficiency for 4-SU. The lower crosslinking efficiency will result in a higher background of sequences derived from fragments of abundant cellular RNAs and therefore you should consider scaling up the experiment when using less efficient photoreactive nucleosides.

    The left panel of Figure 1 shows a comparison of using different photoreactive uridine analogs that could be potentially used for PAR-CliP compared to traditional UV 254 nm crosslinking.

    The intensity of the radioactive band of the correct length gives you a good idea whether the PAR-CliP experiment has worked and you have isolated sufficient RNA to carry through a small RNA sequencing protocol (step-by-step description for cDNA library preparation of small RNAs sequencing can be found in 19). The frequency of characteristic mutations in the sequenced reads, T to C transitions when using 4-SU and G to A transitions when using 6-SG, are indicative of successfully crosslinked sequences. In our experience uncrosslinked RNAs labeled with 4-SU show a background mutation rate of approximately 20%. This rate is increases to approx. 50-80% upon crosslinking.

    Disclosures

    T.T. is a cofounder and scientific advisor to Alnylam Pharmaceuticals and an advisor to Regulus Therapeutics.

    Acknowledgements

    We thank members of the Tuschl laboratory for helpful discussions. M.H. is supported by the Deutscher Akademischer Austauschdienst (DAAD). This work was supported by the Swiss National Fund Grant #3100A0-114001 to M.Z.; T.T. is an HHMI investigator, and work in his laboratory was supported by NIH grants GM073047 and MH08442 and the Starr Foundation.

    Materials

    Buffers and reagents

    Growth medium HEK293 cells

    • DMEM
    • 10% FBS
    • 2 mM L-Glutamine
    • 100 U/ml penicillin
    • 100 U/ml streptomycin
    • 100 μg/ml hygromycin
    • 15 μg/ml blasticidin

    4-thiouridine stock solution (1 M)

    • 260.27 mg 4-thiouridine
    • 1 ml DMSO

    Doxycyclin stock (10 mg/ml)

    • 10 mg doxycyclin
    • 1 ml DMSO

    1x NP40 lysis buffer

    Prepare a stock of 5x buffer without DTT and protease inhibitors. Add DTT and protease inhibitor directly before the experiment.

    • 50 mM HEPES, pH 7.5
    • 150 mM KCl
    • 2 mM EDTA
    • 1 mM NaF
    • 0.5% (v/v) NP40
    • 0.5 mM DTT
    • complete EDTA-free protease inhibitor cocktail (Roche)

    Citrate-Phosphate buffer

    • 4.7 g/l citric acid
    • 9.2 g/l Na2HPO4
    • pH 5.0

    IP-wash buffer

    • 50 mM HEPES-KOH, pH 7.5
    • 300 mM KCl
    • 0.05% (v/v) NP40
    • 0.5 mM DTT (add directly before experiment)
    • complete EDTA-free protease inhibitor cocktail (Roche) (add directly before experiment)

    High-salt wash buffer

    • 50 mM HEPES-KOH, pH 7.5
    • 500 mM KCl
    • 0.05% (v/v) NP40
    • 0.5 mM DTT (add directly before the experiment)
    • complete EDTA-free protease inhibitor cocktail (Roche) (add directly before the experiment)

    Dephosphorylation buffer

    • 50 mM Tris-HCl, pH 7.9
    • 100 mM NaCl
    • 10 mM MgCl2
    • 1 mM DTT

    Phosphatase wash buffer

    • 50 mM Tris-HCl, pH 7.5
    • 20 mM EGTA
    • 0.5% (v/v) NP40

    Polynucleotide kinase (PNK) buffer without DTT

    • 50 mM Tris-HCl, pH 7.5
    • 50 mM NaCl
    • 10 mM MgCl2

    PNK buffer

    • 50 mM Tris-HCl, pH 7.5
    • 50 mM NaCl
    • 10 mM MgCl2
    • 5 mM DTT

    SDS PAGE loading buffer

    • 10% glycerol (v/v)
    • 50 mM Tris-HCl, pH 6.8
    • 2 mM EDTA
    • 2% SDS (w/v)
    • 100 mM DTT
    • 0.1% bromophenol blue

    2x Proteinase K buffer

    • 100 mM Tris-HCl, pH 7.5
    • 150 mM NaCl
    • 12.5 mM EDTA
    • 2% (w/v) SDS

    References

    1. Keene, J. D., RNA regulons: coordination of post-transcriptional events. Nat. Rev. Genet 8 (7), 533 (2007).
    2. Tenenbaum, S. A. et al., Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. Proc. Nat. Acad. Sci. 97 (26), 14085 (2000).
    3. Gerber, A. P. et al., Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster. Proc. Nat. Acad. Sci. 103 (12), 4487 (2006).
    4. Lopez de Silanes, I. et al., Identification of a target RNA motif for RNA-binding protein HuR. Proc. Nat. Acad. Sci. 101 (9), 2987 (2004).
    5. Greenberg, J. R., Ultraviolet light-induced crosslinking of mRNA to proteins. Nucl. Acids Res. 6 (2), 715 (1979).
    6. Wagenmakers, A. J. et al., Cross-linking of mRNA to proteins by irradiation of intact cells with ultraviolet light. Eur. J. Biochem. 112 (2), 323 (1980).
    7. Mayrand, S. et al., Structure of nuclear ribonucleoprotein: identification of proteins in contact with poly(A)+ heterogeneous nuclear RNA in living HeLa cells. The Journal of Cell Biology 90 (2), 380 (1981).
    8. Dreyfuss, G. et al., Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies. Mol. Cell. Biol. 4 (6), 1104 (1984).
    9. Adam, S. A. and Dreyfuss, G., Adenovirus proteins associated with mRNA and hnRNA in infected HeLa cells. J. Virol. 61 (10), 3276 (1987).
    10. Ule, J. et al., CLIP identifies Nova-regulated RNA networks in the brain. Science 302 (5648), 1212 (2003).
    11. Licatalosi, D. D. et al., HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456 (7221), 464 (2008).
    12. Yeo, G. W. et al., An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells. Nat. Struct. Mol. Biol. 16 (2), 130 (2009).
    13. Sanford, J. R. et al., Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts. Genome Res. 19 (3), 381 (2009).
    14. Granneman, S. et al., Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs. Proc. Nat. Acad. Sci. (2009).
    15. Guil, S. and Caceres, J. F., The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a. Nat. Struct. Mol. Biol. 14 (7), 591 (2007).
    16. Chi, S. W. et al., Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460 (7254), 479 (2009).
    17. Zisoulis, D. G. et al., Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans. Nat. Struct. Mol. Biol. advance online publication (2010).
    18. Hafner, M. et al., Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell manuscript accepted for publication (2010).
    19. Hafner, M. et al., Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. Methods 44 (1), 3 (2008).
    20. Andrus, A. and Kuimelis, R. G., Base composition analysis of nucleosides using HPLC. Current Protocols in Nucleic Acid Chemistry Chapter 10, Unit 10 6 (2001).

    Comments

    36 Comments

    great for learning
    Reply

    Posted by: AnonymousJuly 19, 2010, 6:37 PM

    Dear Researchers,

    Thank you for an excellent paper and presentation on your PAR-CLIP procedure. We are attempting to use your method on some of our own research questions.
    A question regarding the crosslinking. The protocol asks for 0.15J/cm². We are assuming the cm² refers to the area of cells in the Petri dish, which for a 15cm plate is 177cm². For crosslinking, that requires ²6.55 J (²65500 uJx100), which far exceeds the capacity of the Stratalinker.
    Would you be so kind as to provide further details for the crosslinking procedure. I thank you for your time.

    Sincerely,

    Craig Tomlinson
    Dartmouth Hitchcock Medical Center
    Reply

    Posted by: AnonymousAugust 6, 2010, 2:34 PM

    Hi Craig,

    I'm not from the Tuschl lab but I noticed your question (it was one I previously had myself). If you look in the stratalinker ²400 manual, they state the units of energy is actually in j/cm², despite what it says on the front of the machine. So your good to go.
    Good hunting,
    MW
    Reply

    Posted by: AnonymousNovember 21, 2011, 10:50 PM

    How stable if the RNA-RNA and RNA-Protein photocrosslinking? Is it possible to denature tU-A crosslinking?
    Reply

    Posted by: AnonymousDecember 11, 2010, 2:15 PM

    The RNA-protein interaction in all CLIP procedures irreversibly creates a covalent bond between RNA and protein. You will not be able to reverse it.
    Reply

    Posted by: AnonymousDecember 13, 2010, 4:46 PM

    why do we need to do dephosphorylation after IP
    Reply

    Posted by: AnonymousApril 3, 2011, 3:22 PM

    It appears that in #² under "Expanding Cells" there is an important typo ... it says to dilute the 4-SU 1:1000 from a 1M stock to get 100uM ... that's not possible. A 1:1000 dilution would be 1,000uM, but to get a 100uM working concentration you would have to do a 1:10,000 dilution. I presume that the error is in the dilution factor because the video makes it explicit that the final concentration should be 100uM. Could someone confirm this for me, either here in the comments or by sending me an email? Thanks!
    Reply

    Posted by: AnonymousNovember 2, 2011, 7:15 PM

    It appears that there is indeed a typo. The concentration should be 100 µM (even though 1 M also shows no toxicity - however, we did not check the incorporation rate) and the appropriate dilution from a 1 M stock is 1:10,000.
    Reply

    Posted by: Markus H.November 7, 2011, 2:29 PM

    Thanks for getting back to me so quickly! I just finished my first PAR-CLIP experiment and have RNA in hand. Two more questions:

    1) Your ²008 Hafner reference for making a cDNA library uses a starting pool of RNAs that is different from that in PAR-CLIP because there should be phosphates at both the 5' and 3' ends in PAR-CLIP (based on the way RNase T1 cuts and the use of T4 PNK to add a radiophosphate to the 5'). The Hafner ²008 protocol uses 5' -Phosphate and 3' hydroxyl, so what adapters or modified protocol did you use for the PAR-CLIP RNAs with phosphates at both ends...?

    ²) What was the size range of RNA fragments/sequence reads from the library? From what I gather in your Cell paper it ranged from ²3-35 nucleotides, but I'm not sure if that's a limit of the sequence/cycle length...
    Reply

    Posted by: AnonymousNovember 30, 2011, 5:12 PM

    Hi Matthew,
    1.) there is no 3'P after CIP treatment (this is also the reason for the CIP). It is also important to chase the hot PNK labeling with cold ATP to make sure that every 5' end is phosphorylated.
    ².) I usually cut out everything between 19 and ~50 nt. When you try the experiment and run the recovered RNA on a gel you will find that the majority will be of ~²0-²5 nt length - hence the size distribution after sequencing.

    I hope this helps.
    Reply

    Posted by: Markus H.December 1, 2011, 10:08 AM

    Thanks for your quick reply, though I'm still a bit confused. 1) I thought CIP removes phosphates only at 5' end of ssRNA, and PNK would add phosphates only to the 5' end ... ? Can you tell me the phospho/hydroxy status of all RNAs at the end of a PAR-CLIP RNA extraction just to be clear? ²) When you say "cut out everything" you mean after the addition of the adapters when you purify via PAGE, right? (sorry for all the questions, I'm just not sure how to proceed to library making based on the status of the RNA fragments at the end of PAR-CLIP)
    Reply

    Posted by: AnonymousDecember 1, 2011, 1:15 PM

    Just checked with NEB and you're right: CIP removes phosphates at both 5' and 3' ends, and T4 PNK adds phosphates to the 5' end and removes any leftover phosphoryl groups at the 3' end. So the resulting RNAs should be 5' phospho and 3' hydroxyl!
    Reply

    Posted by: AnonymousDecember 1, 2011, 1:41 PM

    Yes - this is the way the cDNA preparation protocol is supposed to work.

    Posted by: Markus H.December 1, 2011, 4:00 PM

    Hi Markus,
    I have one question about cutting out the band. You mentioned you usually cut out between 19 and ~50 nt, that is between ~6 kDa and ~17 kDa. Supposing my protein of interest is ~40 kDa, do I need to cut between 46 and 57 kDa?
    Thanks,
    Nian

    Posted by: Nian L.April 26, 2013, 4:06 PM

    Upon delivery the Stratalinker is equipped with ²54nm bulbs; when changing the bulbs to 365nm did you re-calibrate the device? Or did you just trust it anyways and enter 1500 uJ/cm²x100? Measuring with an external UV-meter leads to highly different exposure times than using this setting (exposure would just be around 4-8 seconds in this case). Do you remember how long the exposure time normally is?
    Thanks
    Reply

    Posted by: AnonymousJanuary 5, 2012, 12:53 PM

    When reequipping the Stratalinker with 365 nm bulbs it has to be recalibrated by the company.
    Also take care not to obscure the UV detector with the trays holding the samples. With us the irradiation time is somewhere around ² mins.
    Hope this helps.
    Reply

    Posted by: Markus H.January 5, 2012, 3:03 PM

    Hi, I have a stupid question. The eluted RNA-protein complex is radioactive. Will there be residual radioactivity carried over to the cDNA library that might jeopardize my ability to send samples to outside vendor for high throughput sequenicing?
    Reply

    Posted by: AnonymousFebruary 23, 2012, 12:49 AM

    The RNA gets hydrolyzed before PCR (or during the PCR) and the PCR product purified on a gel, so no. There should not be any radioactivity left in the sample.
    Reply

    Posted by: Markus H.February 23, 2012, 11:56 AM

    Hi, Do you have any good antibody to recommend as a positive control?

    R
    Reply

    Posted by: AnonymousMarch 6, 2012, 12:12 PM

    Hi R,
    we have successfully used the AGO² antibody (Millipore, http://www.millipore.com/catalogue/item/04-64²).
    There was a problem with one lot number, but this should have been corrected.
    Reply

    Posted by: Markus H.March 12, 2012, 12:08 PM

    Hi, I was wondering why the RNaseT1 digest is done at ²²C and not 37C? Thanks.
    Reply

    Posted by: Priya B.June 11, 2012, 4:31 PM

    Hi,

    We were looking into purchasing a stratalinker for Clip and northern blot analyses. However, Stratagene was bought out and the stratalinker has been discontinued. DŒs anyone know of a recommendable alternative?

    Thank you,
    LR
    Reply

    Posted by: Labib R.July 12, 2012, 6:13 PM

    I've been trying to run experiments with 4-SU and 365nm crosslinking, but it seems that the crosslinking efficiency is not good and I do not know what else to troubleshoot. Do you have any advice? I saw above that it took about ² minutes for you to crosslink. My crosslinker dŒsn't take that long; should I extend the time to ensure better results? Is the distance from the bulbs important? Is there something special about the 4-SU (sigma) before adding it to your cells? Sorry for the simple question, but I'm out of ideas.
    -Matt
    Reply

    Posted by: AnonymousSeptember 17, 2012, 6:27 PM

    The Sigma 4SU should be fine. But I would suggest using a positive control - e.g. HEK²93 and compare uptake via HPLC (as described in the various Methods publications) - or check the intensity of a PAR-CLIP band for an RBP for which a good antibody is available. Check IP efficiency. If you find out that the incorporation rate of 4SU is low, vary the 4SU concentration (1 mM is still fine e.g. in HEK²93) - you can also vary the incorporation time; if the cells proliferate very rapidly, the 4SU may already be used up in the overnight labeling.
    It appears that insect cells have a hard time taking up 4SU - it might help to split them directly into the $SU containing medium. Yeast are able to take up 4-thiouracil (the base itself).

    Just a number of suggestions off the top of my head.
    Reply

    Posted by: Markus H.September 17, 2012, 7:18 PM

    Another quick question: did you just buy 4-SU from Sigma and add it directly to the cells? I see that it has to be phosphorylated before it can be incorporated into RNA. DŒs this happen in vivo after uptake?
    Reply

    Posted by: AnonymousSeptember 18, 2012, 3:17 PM

    4SU from Sigma worked perfectly fine for us. All nucleoside analogues have to be triphosphorylated before they can be incorporated into nascent RNA. This happens after uptake. Note that triphosphorylated and therefore highly charged compounds cannot cross the membrane.
    Reply

    Posted by: Markus H.September 18, 2012, 7:14 PM

    I did a miseq run with an Ago² precipitated sample and see that 40% of my reads are clusters of "As". Not sure why. According to the RNase T1 that I checked by radioactivity, it looked like it gave me a good band on the gel which I purified and then proceeded with the experiment. So it should not be polyadenylated RNA. Have any of you seen anything like this?
    Reply

    Posted by: Go G.November 1, 2012, 7:20 PM

    check with your sequencing expert. It may be that the miseq software reports an A by default if there is no signal in the cycle. This means that your inserts were very short - and then the software added the A's after sequencing out of the adapter.
    Reply

    Posted by: Markus H.November 2, 2012, 12:36 PM

    What percentage of your reads in a Hiseq sequencing are expected to align to human? Is there noise due to increased RNAseT1?
    Reply

    Posted by: Go G.December 12, 2012, 7:13 PM

    the fraction of reads mapping to the genome depends on 1.) the amount of RNA recovered and ².) on the purity of the RNA ligase enzymes used in the cDNA preparation. if they contain bacterial RNA sequences you can get a substantial (up to 70% or more) fraction of sequences mapping to bacterial rRNA.
    Reply

    Posted by: Markus H.December 17, 2012, 9:18 AM

    Hi Dr.
    I am Renata Romero, I'm from Mexico City and I wonder if this technique can be implemented in tissues.

    Thank you.

    Best regards
    Reply

    Posted by: RENATA R.February 26, 2013, 8:06 PM

    You need to find a way to get 4SU into the tissues - at least for worms that is possible (Jungkamp et al., Mol. Cell, ²01²). Otherwise you will have to rely on another method, such as iCLIP.
    Reply

    Posted by: Markus H.February 27, 2013, 1:31 PM

    Hello, could you please explain why there is a high-salt wash step at 500mM KCl? Thank you!
    Reply

    Posted by: Al A.March 27, 2013, 6:32 PM

    Hello,

    Could you help me with some problems?
    1. I am wondering what control (IgG coupled beads or just beads only) did you use for PAR-CLIP and further bioinformatics analysis?
    ². Why you choose small RNA library but custom cDNA library. I want to purify pull-downed RNAs after second RNase T1 for cDNA library construction and RNA-seq. So the steps for RNA end modificatin could be omitted. Do you think it is feasible?

    Thanks very much.
    Reply

    Posted by: Xu Z.May 10, 2013, 6:46 AM

    1. Please read the review by Ascano et al in WIRes RNA (²01²) - there we discuss the issues with the controls.
    ². The standard RNAseq cDNA library construction of course never works for small RNAs. Here you need to go for small RNA cDNA construction.
    Reply

    Posted by: Markus H.May 10, 2013, 8:43 AM

    Hi
    I'm a student and I'm interested in this technique
    I really can't figure out why the T-C transitions take place. They are obviously directly caused by the photoactivatable ribonucleoside analog used but I don't understand the molecular mechanism that leads to the transition. In particular I don't see the conncetion between the crosslinking and the transition; given that at the time of the retrotrascription, the samples were already been treated with K proteinase and so the crosslinking gone.
    Reply

    Posted by: marco n.July 8, 2013, 4:54 PM

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