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Chromatin Isolation by RNA Purification (ChIRP)

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1Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine

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    Summary

    ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

    Date Published: 3/25/2012, Issue 61; doi: 10.3791/3912

    Cite this Article

    Chu, C., Quinn, J., Chang, H. Y. Chromatin Isolation by RNA Purification (ChIRP). J. Vis. Exp. (61), e3912, doi:10.3791/3912 (2012).

    Abstract

    Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.

    Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

    Protocol

    1. Probe Design

    Design anti-sense DNA tiling probes for selective retrieval of RNA target by ChIRP.

    1. Design anti-sense oligo probes using the online probe designer at singlemoleculefish.com 18.
    2. Use these parameters: number of probes = 1 probe /100 bp of RNA length; 2) Target GC% = 45; 3) Oligonucleotide length = 20; 4) Spacing length = 60-80. Break RNA into segments if too long for the designer. Omit regions of repeats or extensive homology.
    3. Order anti-sense DNA probes with BiotinTEG at 3-prime end.
    4. Label probes according to their positions along the RNA. Separate them into two pools so that the "even" pool contains all probes numbering 2, 4, 6, etc. and the "odd" pool contains probes numbering 1, 3, 5, etc. Dilute pool of probes to 100 μM concentration and store at -20 °C.
    5. All experiments are to be performed using both pools, which serve as internal controls for each other. Real RNA-dependent signal would be present from both pools, while probe-specific noises would be unique to each pool. This applies for both ChIRP-qPCR and ChIRP-seq.

    2. Harvest Cells

    Collect cells that will be used for ChIRP experiment.

    1. Grow cells in tissue culture plates or flasks to confluency Rinse with phosphate buffered saline (PBS) once and trypsinize Quench trypsin with >2x volume of media, pipette up and down to dislodge cells and resuspend into single cell suspension. Transfer all media and resuspended cells into 50 ml Falcon tubes. 20 million cells are typically sufficient for one ChIRP sample.
    2. Spin cells at 800RCF for 4 min. Aspirate media and resuspend 40 million cells in 40 ml of PBS, combine tubes if necessary. Spin cells at 800RCF for 4 min. Decant PBS, carefully aspirate on an angle the remaining liquid.

    3. Cross-link Cells and Collect Cell Pellet

    Crosslink collected cells with glutaraldehyde to preserve RNA-Chromatin interactions and prepare cell pellet.

    1. Perform all steps at room temperature.
    2. Prepare 1% glutaraldehyde in room temperature PBS. Prepare 10 ml per 10 million cells (0.4 mL 25% glutaraldehyde stock + 9.6 mL PBS). Glutaraldehyde must be used fresh.
    3. Tap bottom of Falcon tubes to dislodge pellets. Resuspend cell pellet in 1% glutaraldehyde, starting with a small volume to avoid chunks, then top up to full volume. Invert to mix. Crosslink for 10min at room temperature on an end-to-end shaker or rotator.
    4. Quench the cross-linking reaction with 1/10th volume of 1.25 M glycine at room temperature for 5 min.
    5. Spin at 2000RCF for 5 min. Aspirate supernatant and wash pellet with 20 mL chilled PBS once, spinning at 2000RCF for 5 min.
    6. Aspirate and resuspend the washed, cross-linked pellet with 1 ml chilled PBS per 20 million cells. Transfer each ml to an Eppendorf tube and spin at 2000RCF for 3 min at 4 C. Remove as much PBS as possible with pipette tip carefully.
    7. Flash-freeze the cell pellets in liquid nitrogen and store at -80 °C indefinitely.

    4. Cell Lysis

    Lyse crosslinked cells to prepare cell lysate.

    1. Thaw frozen cell pellets at room temperature. Tap hard to dislodge and mix the cell pellet. Spin down the pellet at 2000RCF for 3 min at 4 °C. Use a sharp 10 μl pipette tip to remove any remaining PBS.
    2. On an electronic balance (accurate to 1mg) tare the mass of an empty Eppendorf tube (our tubes weigh 1.060 grams very consistently). Weigh each pellet and record its weight. A full 15 cm dish of crosslinked HeLa cells typically weighs 100 mg.
    3. Supplement Lysis Buffer (10X the mass of pellet, e.g. 1 ml for 100mg) with fresh Protease Inhibitor, PMSF and Superase-in (see attached buffer list). Mix well.
    4. Add 10X volume supplemented Lysis Buffer to each tube and resuspend the pellet. For small pellets <25 mg, resuspend in 250 μl supplemented lysis buffer Suspension should be smooth. If not, divide suspension into 500 μl aliquots and use a motorized pellet mixer to break up clumps. Proceed immediately to sonication.

    5. Sonication

    Shear DNA by sonicating crosslinked cell lysates.

    1. Sonicate cell lysate in Bioruptor in 15 ml Falcon tubes. Use <1.5 ml lysate in each tube, and for faster sonication, sonicate no more than two tubes at a time.
    2. Sonicate in a 4 °C water bath at highest setting with 30 seconds ON, 45 seconds OFF pulse intervals. Check lysate every 30 min. Continue sonicating until the cell lysate is no longer turbid. This may take as little as 30 min and as many as 4 hrs. The number of tubes, the sample volume, the bath temperature, and the period of sonication time will affect how long the process takes. Tubes will likely sonicate at different rates, so pool them together every 30 min and redistribute into original tubes to ensure homogeneity. Note: glutaraldehyde-crosslinked cells take significantly longer to sonicate than formaldehyde equivalents.
    3. When lysate turns clear, transfer 5 μL lysate to a fresh Eppendorf tube. Add 90 μL DNA Protease K (PK) Buffer (see buffer list) and 5 μL PK. Vortex to mix and spin down briefly. Incubate for 45 min at 50 °C.
    4. Extract DNA with Qiagen PCR purification kit. Elute DNA in 30 μL Qiagen Elution Buffer (EB) and check DNA size on 1% agarose gel. If bulk of the DNA smear is 100-500 bp, sonication is complete. If not, continue to sonicate.
    5. Centrifuge sonicated samples at 16100RCF for 10 min at 4 °C. Combine supernatants, aliquot into 1 mL samples and flash-freeze in liquid nitrogen. Store at -80 °C.

    6. ChIRP

    Hybridize biotinylated DNA probes to RNA and isolate bound chromatin.

    1. Thaw tubes of chromatin at room temperature.
    2. Prepare Hybridization Buffer (see buffer list, prepare 2 ml per ml of chromatin). Vortex to mix.
    3. For a typical ChIRP sample using 1 ml of lysate, remove 10 μL for RNA INPUT and 10 μL for DNA INPUT and place in Eppendorf tubes. Keep on ice till further use.
    4. Transfer 1 mL chromatin to 15 mL Falcon tube. Add 2 mL Hybridization Buffer to each tube. For total volume <1.5 ml, use Eppendorf tubes.
    5. Thaw probes at room temperature. Nanodrop probes to check amount if you haven't used it in a long time (100 μM probes should spec ~500-600 ng/μl using single strand DNA setting). Add appropriate volume of probes to specific tubes (100 pmol probe per 1 mL chromatin, 1 μL of 100 pmol/μL probe per 1 mL chromatin). Mix well. Incubate at 37 °C for 4 hrs with shaking.
    6. With 20 min remaining for hybridization, prepare the C-1 magnetic beads (stored at 4 °C). Use 100 μL per 100 pmol of probes. Wash with 1 mL unsupplemented Lysis Buffer three times, using the DynaMag-2 magnet strip to separate beads from buffer.
    7. Resuspend beads in original volume of Lysis Buffer; supplement with fresh PMSF, P.I and Superase-in. After 4 hr hybridization reaction is complete, add 100 μL beads to each tube. Mix well. Incubate at 37 °C for 30 min with shaking.
    8. Prepare Wash Buffer (5 mL per sample). Vortex to mix. Pre-warm to 37 °C. Add PMSF before use.
    9. Wash beads with 1 mL wash buffer five times. On the first wash, use DynaMag-15 magnetic strip to separate beads, decant, and resuspend in 1 mL wash buffer. Transfer volume to 1.5 mL Eppendorf tube. Incubate at 37 °C with shaking for 5 min.
    10. On subsequent washes, spin down each tube on a minicentrifuge, set sample on DynaMag-2 magnetic strip for 1 min. Decant sample, wipe any drips with a Kimwipe, resuspend in 1 mL wash buffer. Incubate at 37 °C with shaking for 5 min. Repeat for five total washes.
    11. At last wash, resuspend the beads well. Remove 100 μL and set aside for RNA isolation Reserve 900 μL for DNA fraction. Place all tubes on DynaMag-2 magnetic strip and remove wash buffer. Spin all tubes down briefly; place them on magnet strip. Remove the last bit of wash buffer completely with a sharp 10 μl pipette tip.

    7. RNA Isolation

    Extract RNA fraction from ChIRP samples to quantitate by qRT-PCR.

    1. Take 100 μL bead samples and 10 μL RNA INPUT sample. Add 85 μL RNA PK Buffer pH 7.0 to RNA INPUT. Resuspend beads in 95 μL RNA PK Buffer pH 7.0. Add 5 μL Proteinease K and incubate at 50 °C for 45 min with end-to-end shaking.
    2. Briefly spin down all tubes and boil samples for 10 min on heat block at 95 °C.
    3. Chill samples on ice, add 500 μL TRIzol, vortex vigorously for 10 sec. Incubate at room temperature for 10 min. Store at -80 °C or proceed to step 4.
    4. Add 100 μL chloroform to TRIzol treated samples. Vortex vigorously for 10 sec. Spin at 16100RCF on a benchtop centrifuge for 15 min at 4 °C.
    5. Remove ~400 μL aqueous supernatant, avoiding organic and interface.
    6. Add 600 μL (1.5 volume) 100% ethanol and mix well. Spin sample through MIRNeasy mini columns. Wash 1x with RWT (MIRNeasy mini kit), 2x with RPE per manufacturer's protocol. Elute with 30 μL nuclease-free H2O (nfH2O).
    7. Treat the RNA eluate with DNA-free per manufacturer's protocol. After the reaction is complete, heat the sample for 15 minutes at 65 °C to completely inactivate any remaining DNase.
    8. Use 1 μL RNA isolate per well for qRT-PCR analysis to confirm lncRNA retrieval. GAPDH is often used as a negative control.

    8. DNA Isolation

    Extract DNA fraction from ChIRP samples to identify by sequencing or quantitate by qPCR.

    1. Prepare DNA Elution Buffer (see buffer list), 150 μl per sample, including DNA INPUT.
    2. Add 1 0μL RNase A (10 mg/mL) and 10 μL RNase H (10 U/μl) per ml of DNA Elution Buffer, and vortex to mix.
    3. Resuspend each sample of beads in 150 μL of DNA Elution Buffer with RNases. (Resuspend DNA INPUT in 140 μL) Incubate at 37 °C for 30 min with shaking.
    4. Separate beads and supernatant on DynaMag-2 magnetic strip. Remove supernatant and add to labeled tubes.
    5. Prepare a second aliquot of DNA Elution Buffer with 10 μL RNase A (10 mg/mL) and RNaseH (10 U/μL) exactly as done in 8.2). Add 150 μL to each sample (including DNA INPUT), incubate, and remove supernatant. Collect all supernatant (should be ~300 μL).
    6. Add 15 μL PK to each sample. Incubate at 50 °C for 45 min with shaking.
    7. Pre-spin down yellow phase-lock gel tubes (5PRIME). Transfer DNA samples to phase-lock gel tubes, and add 300 μL PhOH:Chloroform:Isoamyl per sample. Shake vigorously for 10 min, and spin down on a benchtop centrifuge at 16100RCF for 5 min at 4 °C. Take aqueous from the top (~300 μL). Add 3 μL GlycoBlue, 30 μL NaOAc, and 900 μL 100% EtOH. Mix well and store at -20 °C overnight.
    8. Spin samples at 16100RCF for 30 min at 4 °C.
    9. Decant supernatant carefully. Add 1 mL 70% EtOH and vortex to mix. Spin at 16100RCF for 5 min. Remove supernatant by pipette. Air dry for 1min. Resuspend in 30 μL EB.
    10. DNA samples are ready for analysis by qPCR or preparation of high-throughput sequencing libraries per Illumina protocol.

    10. Representative Results

    Figure 1 depicts the ChIRP workflow. A successful ChIRP experiment typically enriches target RNA significantly over non-specific RNAs. Figure 2 shows enrichment of human telomerase RNA (TERC) from HeLa cells over GAPDH, an abundant cellular RNA that serves as a negative control. Majority of TERC RNAs (~88%) present in the cell were pulled down by performing ChIRP, whereas only 0.46% of GAPDH RNA was retrieved, demonstrating an enrichment factor of ~200 fold. Nonspecific probes such as probes targeting LacZ RNA, which is not expressed in mammalian cells (Figure 2), can be used as additional negative controls.

    DNA regions expected to bind the target lncRNA are typically enriched over negative regions when measured by qPCR. Figure 3 shows qPCR validation of four HOTAIR-bound sites in primary human foreskin fibroblasts that we determined by performing ChIRP-seq in the same cell line, while TERC and GAPDH DNA sites serve as negative control regions. Both "even" and "odd" probe sets yielded comparable enrichment of expected HOTAIR-bound sites over negative regions, a hallmark of true lncRNA-binding sites.

    High-throughput sequencing of ChIRP enriched DNA yields a global map of lncRNA-binding sites. The Drosophila lncRNA roX2 is known to interact with the X-chromosome in a manner that is required for dosage compensation. Figure 4 shows roX2 binding profile over a section of the X chromosome. Both "even" and "odd" samples have been sequenced and their unique noises have been eliminated to produce a track of overlapping signals. Each "peak" here indicates a strong site of roX2 binding. The complete track and list of roX2 target genes have been described in Chu et al. 2011 17.

    Figure 1.
    Figure 1. Flow chart of the ChIRP procedure. Chromatin is crosslinked to lncRNA:protein adducts in vivo. Biotinylated tiling probes are hybridized to target lncRNA, and chromatin complexes are purified using magnetic streptavidin beads, followed by stringent washes. We elute lncRNA bound DNA or proteins with a cocktail of Rnase A and H. A putative lncRNA binding sequence is schematized in orange. Previously published in Chu et al. 2011.17

    Figure 2.
    Figure 2. ChIRP enriches for human TERC RNA. TERC-asDNA probes retrieve ~88% of cellular TERC RNA and undetectable GAPDH. LacZ-asDNA probes are used as negative controls and retrieve neither RNAs. Mean + s.d. are shown. Previously published in Chu et al. 2011.17

    Figure 3.
    Figure 3. HOTAIR ChIRP-qPCR in primary human foreskin fibroblasts. NFKBIA, HOXD3-4, SERINC5 and ABCA2 are regions that interact with HOTAIR. TERC and GAPDH served as negative controls. Mean + s.d. are shown. Previously published in Chu et al. 2011.17

    Figure 4.
    Figure 4. ChIRP-seq data of roX2 RNA in Sl2 Drosophila cells. "Even" and "odd" were sequenced separately; their data merge to reflect only common peaks in both. The merged track is shown. Previously published in Chu et al. 2011.17

    Discussion

    Here we described ChIRP-seq, a method of mapping in vivo lncRNA binding sites genome-wide. The key parameters for success are the split pools of tiling oligonucleotide probes and glutaraldehyde crosslinking. The design of affinity-probes is straightforward given the RNA sequence and requires no prior knowledge of the RNA's structure or functional domains. Our success with roX2, TERC, and HOTAIR - three rather different RNAs in two species - suggests that ChIRP-seq is likely generalizable to many lncRNAs. As with all experiments, care and proper controls are required to interpret the results. Different lncRNA may require titration of conditions, and judicious change of conditions, such as selection of different affinity probes or crosslinkers, may highlight different aspects of RNA-chromatin interactions. Like ChIP-seq, not all binding events are necessarily functional, and additional studies are required to ascertain the biological consequences of RNA occupancy on chromatin. Nonetheless, we foresee many interesting applications of this technology for researchers of other chromatin-associated lncRNAs, which now number in the thousands8,9. Just as ChIP-seq has opened the door for genome-wide explorations of DNA-protein interactions, ChIRP-seq studies of the "RNA interactome" may reveal many new avenues of biology.

    Disclosures

    C. Chu and H.Y. Chang are named as inventors on a patent application based on this method.

    Acknowledgements

    We thank T. Hung, MC. Tsai, O. Manor, E. Segal, M. Kuroda, T. Swigut, and I. Shestopalov for discussions. Supported by the Agency of Science, Technology and Research of Singapore (C.C.), NIH R01-CA118750 and R01- HG004361 (H.Y.C.), and California Institute for Regenerative Medicine (H.Y.C.). H.Y.C. is an Early Career Scientist of the Howard Hughes Medical Institute.

    Materials

    Name Company Catalog Number Comments
    Buffer List:
    Dissolve a pellet of complete protease inhibitor in 1 ml water as 50x stock. Make 100 mM PMSF in isopropanol (100x stock). Superase-in is used as 200x stock. Store all at -20 °C.
    Lysis Buffer:
    50 mM Tris-Cl pH 7.0
    10 mM EDTA
    1% SDS
    Always add PMSF, P.I. and Superase-in fresh before use except when washing beads
    Proteinase K Buffer (for DNA)
    100 mM NaCl
    10 mM TrisCl pH 8.0 (For RNA use pH 7.0)
    1 mM EDTA
    0.5% SDS
    Add 5% by volume Proteainse K (Ambion AM2546 20 mg/ml) fresh before use
    Hybridization Buffer
    750 mM NaCl
    1% SDS
    50 mM Tris-Cl pH 7.0
    1 mM EDTA
    15% formamide (store in the dark at 4 °C)
    Always add PMSF, P.I. and Superase-in fresh before use
    Wash Buffer
    2x NaCl and Sodium citrate (SSC) (diluted from 20x SSC Invitrogen stock)
    0.5% SDS
    Always add PMSF fresh before use
    DNA elution Buffer
    50 mM NaHCO3
    1% SDS
    Table of specific reagents and equipment:
    Glutaraldehyde (EM grade) Sigma-Aldrich G5882-10x10ml
    Motorized pellet mixer VWR international V8185-904
    Protease inhibitor Roche Group 11873580001
    PMSF Sigma-Aldrich 78830
    Superase-in Ambion AM2696
    Bioruptor Diagenode UCD-200
    Falcon tubes (for sonication) Corning 430790
    Proteinase K Ambion AM2546
    PCR purification kit Qiagen 28106
    C-1 magnetic beads Invitrogen 65002
    PMSF Sigma-Aldrich P7626-25G
    DynaMag-15 magnet Invitrogen 123-01D
    DynaMag-2 magnet Invitrogen 123-21D
    MIRNeasy mini kit Qiagen 217004
    Rnase H Epicentre Biotechnologies R0601K
    Rnase A Sigma-Aldrich R4875-100MG
    Phase Lock Gel Heavy 5 PRIME 2302810
    Trizol Invitrogen 15596-018
    Phenol:chloroform:Isoamyl Invitrogen 15593-031
    Chloroform Ricca RSOC0020-1C
    GlycoBlue Ambion AM9515
    Glycine JT Baker 4057-06
    PBS, pH 7.4 Invitrogen 10010-049
    Elution Buffer (EB) Qiagen 19086
    20x SSC Invitrogen 15557-036
    10% SDS Invitrogen 15553-027
    DNA-free Ambion AM1906
    Buffer kit Ambion AM9010
    Formamide Invitrogen 15515-026

    References

    1. Koziol, M. J., Rinn, J. L. RNA traffic control of chromatin complexes. Curr. Opin. Genet. Dev. 20, 142-148 (2010).
    2. Mercer, T. R., Dinger, M. E., Mattick, J. S. Long non-coding RNAs: insights into functions. Nat. Rev. Genet. 10, 155-159 (2009).
    3. Rinn, J. L. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell. 129, 1311-1323 (2007).
    4. Zhao, J., Sun, B. K., Erwin, J. A., Song, J. J., Lee, J. T. Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science. 322, 750-756 (2008).
    5. Kelley, R. L. Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin. Cell. 98, 513-522 (1999).
    6. Pandey, R. R. Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation. Mol. Cell. 32, 232-246 (2008).
    7. Wang, K. C. A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature. 472, 120-124 (2011).
    8. Khalil, A. M. Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc. Natl. Acad. Sci. U.S.A. 106, 11667-11672 (2009).
    9. Zhao, J. Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol. Cell. 40, 939-953 (2010).
    10. Meller, V. H., Wu, K. H., Roman, G., Kuroda, M. I., Davis, R. L. roX1 RNA paints the X chromosome of male Drosophila and is regulated by the dosage compensation system. Cell. 88, 445-457 (1997).
    11. Franke, A., Baker, B. S. The rox1 and rox2 RNAs are essential components of the compensasome, which mediates dosage compensation in Drosophila. Mol. Cell. 4, 117-122 (1999).
    12. Gupta, R. A. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature. 464, 1071-1076 (2010).
    13. Tsai, M. C. Long noncoding RNA as modular scaffold of histone modification complexes. Science. 329, 689-693 (2010).
    14. Zappulla, D. C., Cech, T. R. RNA as a flexible scaffold for proteins: yeast telomerase and beyond. Cold Spring Harb. Symp. Quant. Biol.. 71, 217-224 (2006).
    15. Nagano, T. The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin. Science. 322, 1717-1720 (2008).
    16. Carter, D., Chakalova, L., Osborne, C. S., Dai, Y. F., Fraser, P. Long-range chromatin regulatory interactions in vivo. Nature. 32, 623-626 (2002).
    17. Chu, C., Qu, K., Zhong, F. L., Artandi, S. E., Chang, H. Y. Genomic Maps of Long Noncoding RNA Occupancy Reveal Principles of RNA-Chromatin Interactions. Mol. Cell. (2011).
    18. Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A., Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods. 5, 877-879 (2008).

    Comments

    78 Comments

    hi,i has a great interest on CHIRP and your works are highly appreciated,but i want to know that where can i find the buffer list?thank you
    Reply

    Posted by: tang n.May 5, 2012, 4:16 AM

    Hi there, thanks for the interest in our paper. As you pointed out the original article lacked the buffer list, which we have uploaded a while ago. Hope it helps!
    Reply

    Posted by: Ci C.December 18, 2012, 5:20 PM

    Hello, I'm trying to perform ChIRP experiments and your protocol is very helpful to me.

    However, I have a question about the final concentration of biotinylated probes and the volume of the beads.
    My probes are 100µM so 1µL corresponds to 100pmol. I have 50 probes and my question is : should I use 100µL of magnetic C1 beads per 100pmol of probe, so in my case, 50 probes x 100µL = 5mL of beads ?

    Thanks a lot for the answer ?
    Reply

    Posted by: Sylvain F.October 31, 2012, 4:47 AM

    I also have this question!
    Reply

    Posted by: Guihai f.November 28, 2012, 7:51 AM

    Hi guys, thanks for the interest in our paper. That's indeed a common question I get, so apologize for not explaining that more clearly in the paper. The 100uM concentration refers to all probes, so if you have ² probes in the pool the individual concentration is 50uM per probe, and if you have 50 probes it's ²uM per probe. It follows that as you scale up number of probes in the pool you do not have to scale up the amount of beads to add. As a caution though, we have noticed that when I use too many probes the concentration of each probe drops too much and it actually adversely affects yield. Try not to use more than 50 probes if possible.
    Reply

    Posted by: Ci C.December 18, 2012, 5:23 PM

    what is the fucntion of the Buffer kit ? I can not find it in the protocol.

    Thanks for your time!
    Reply

    Posted by: Guihai f.November 1, 2012, 12:50 AM

    The buffer kit is simply a convenient collection of buffers provided by Ambion that includes 5M NaCl, 1M Tris, 0.5M EDTA, etc..
    Reply

    Posted by: Ci C.December 18, 2012, 5:25 PM

    Hi, I refer to your paper in HOTAIR ChIRP. You used 48 oligo probes for this ncRNA. I am trying to do a HOTAIR ChIRP. I am wondering if the number of probes can be smaller to reduce cost without a huge compromise on specificity. Furthermore, I am not doing sequencing but a specific check on a genomic region of interest, so I supposed the background noise may be lower? Thank you!
    Reply

    Posted by: Yi Fang L.November 8, 2012, 5:11 AM

    We share your desire to reduce number of probes used. In preliminary testing I've found that you can use as few as 1 probe per ²00 nucleotide of RNA target RNA. But that number has to be doubled considering if you need both "even" and "odd" set, so effectively tiling density is 1 probe / 100nt.
    Reply

    Posted by: Ci C.December 18, 2012, 5:28 PM

    Hello,
    I enjoyed your paper very much.

    At the end of the video, and in the figure, you mention that in this process you can isolate RNA binding proteins that were associated with the RNA. My questions are: have you had success in doing this? What is the protocol for isolating the proteins? Do you just isolate the protein fraction from the Trizol, or is there a more specialized method? Is there a paper that may lay out use of this method?

    Thank you for your help.
    Reply

    Posted by: Gavin J.January 18, 2013, 12:09 PM

    Hi,
    Great protocol. Just one question. You mention that all ChIRP steps have to be performed at 37C. I can imagine this is important for the hybridisation steps, but I was wondering why this was needed for the washing steps as well?

    Many thanks
    Reply

    Posted by: Sebastian V.March 15, 2013, 10:21 AM

    The washing should be performed at 37C just like you would wash a northern blot at elevated temperatures. If this is logistically difficult for you, just warming up the buffer to 37C and washing on a room temp. shaker works well too.
    Reply

    Posted by: Ci C.March 15, 2013, 1:47 PM

    Hello,
    Thanks for the detailed and very helpful protocol. Can you tell me what kind of DNA yields to expect from the pull down per input cell amount? I know it will depend on the lncRNA targeted, but I need an idea of what kind of amounts to expect. Could you tell me what you got for your HOTAIR hybridizations? My own hybs only result in a few nanograms of DNA, and I don't know how to determine whether that is nonspecific DNA binding to my beads or whether it is actual lncRNA-bound DNA? How do you control for this? Thank you for your help in advance.
    Reply

    Posted by: blake e.March 20, 2013, 5:44 AM

    Hi,

    Congrats for setting up such a protocol. It might have been discussed already but I would like to have an idea about the typical amounts of DNA and RNA that one might expect from 20 x 10 ^6 cells. This of course is transcript specific, but should we expect nanograms, picograms for either DNA and RNA?

    Also have you tried FA instead of glutaraldehyde for x-linking? Many thanks for your time
    Reply

    Posted by: Antonis A.November 26, 2013, 12:15 PM

    Hi,

    Thanks for the detailed protocol. I am about to embark on it over the next week or so and had a question regarding the sonication. You advise a Bioruptor for this step however we only have a Covaris available. Would that provide acceptable sonication and do you have any advice regarding appropriate settings?

    Thank you

    Joshua
    Reply

    Posted by: joshua b.March 2, 2014, 12:24 AM

    Yes Covaris works, we've found that it generally reduces sonication time by 30-50% compared with Diagenode. Actual time and intensity needs to be optimized for each machine and cell line.
    Reply

    Posted by: Ci C.March 3, 2014, 5:51 PM

    Hi,

    Thanks for the detailed protocol. I am about to embark on it over the next week or so and had a question regarding the sonication. You advise a Bioruptor for this step however we only have a Covaris available. Would that provide acceptable sonication and do you have any advice regarding appropriate settings?

    Thank you

    Joshua
    Reply

    Posted by: joshua b.March 2, 2014, 6:32 AM

    To all interested ChIRPers, hybridization can be performed overnight instead of 4hours. Doing so greatly reduces hands-on time. Happy ChIRPing!
    Reply

    Posted by: Ci C.March 3, 2014, 8:14 PM

    Hi Ci,

    I have one more issue to resolve regarding ChIRPseq that I would appreciate your input on please (or other ChIRPers that have had similar problems).

    We have been unable to produce lysates using glutaraldehyde despite following the protocol to the letter and getting the Sigma brand that is recommended. Even before sonication, the DNA is degraded to a thick band below 200bp. When we try exactly the same process but substitute 1% formaldehyde (which we normally use for ChIP and 3C) or even plain PBS, the DNA is fine. We have even tried doing everything on ice and reducing the centrifuge steps to only 500g but to no avail.

    My questions therefore are:

    1/ Has your team had similar issues with glutaraldehyde in the past or found it to behave quite differently to formaldehyde (apart from needed longer sonication and harder centrifuging)?
    2/ Though you have shown glutaraldehyde to give a better signal to noise ratio, could we still expect to get usable data using 1% formaldehyde or is it not worth the time and effort?

    Thank you for your time

    Joshua Betts
    Reply

    Posted by: joshua b.April 16, 2014, 8:07 AM

    Dear Joshua,

    Thanks for your interest in our protocol. It's unfortunate to hear that you're experiencing DNA degradation. We've never experienced this issue, nor have we heard similar problems from our collaborators who successfully did ChIRP. Usually we're more concerned with RNA integrity, naturally, and if you're careful that's not a problem either.

    Could you tell me how you're extracting DNA, especially prior to sonication?

    Regarding alternative crosslinking methods, for certain lncRNAs we've found that strong formaldehyde crosslinking also works (3% for 30min is a good starting point). 1% formaldehyde usually does not work.

    Best,
    Chu
    Reply

    Posted by: Ci C.April 16, 2014, 1:55 PM

    Dear Chu,

    Thank you for your advice - I will try the protocol again but substitute 3% formaldehyde for the glutaraldehyde. Does that require any other adjustments apart from slower centrifuge spins and shorter sonication times?

    In regards to my DNA isolation, I lysed the cells as per the protocol and then treated with proteinase K for 45mins (at 50deg or 65deg - either seemed ok). I then RNAse treated the sample and either phenol chloroform extracted the DNA or instead used the Qiagen PCR columns before running the product on a 1% agarose gel. The columns gave more of a smear on the gel, whilst the phenol chloroform extraction just had a broad band below 100bp.

    When I performed exactly the same procedures on formaldehyde treated cells, I obtained a bright band of gDNA at the top of the gel as well as a more faint diffuse band below 100bp. This is what we normally see when working with formaldehyde. Am I right to expect a strong band of genomic DNA on the gel when using glutaraldehyde before sonication, or does the glutaraldehyde break the gDNA down during the fixation process? Does your glutaraldehyde fixed sample normally start as a broad smear even prior to sonication?

    Our group has a lot of experience with ChIPseq, 3C and 4Cseq library preparation but we have never dealt with glutaraldehyde before so are unsure what to expect.

    Any further suggestions would be greatly appreciated.

    Thanks

    Joshua
    Reply

    Posted by: joshua b.April 24, 2014, 9:26 AM

    Before preparing your ChIRP library for sequencing, do you check your DNA size distribution (for example using Agilent Bioanalyzer). If so, do you see a similar distribution to that seen for a typical ChIP sample (200-800 bp range)?
    Reply

    Posted by: Eric S.August 29, 2014, 2:43 PM

    Dear Chu
    I have two questions:
    1.In designing your chirp oligos have you tried to reduce the spacer between biotin-oligonucleotides. Is there any steric reason for choosing 1 probe / 100nt?
    2. Did you tried to crosslink adherent cells directly on plate? we have problem in properly resuspend our differentiated murine myoblasts in PBS+glutaraldehyde.

    thanks in advance
    regards

    Mariangela

    Reply

    Posted by: Mariangela M.October 16, 2014, 9:56 AM

    Dear Mariangela,

    1. It's both a cost consideration (fewer probes are cheaper) and also a biochemical noise consideration (more probes introduce higher chances for probe:DNA direct hybridization).

    2. Yes we've found no difference fixing on plate vs. in suspension. Feel free to pick and choose what works best for you.

    Best,
    Chu
    Reply

    Posted by: Ci C.October 17, 2014, 10:43 PM

    Dear ChIRP community,

    We are having problems getting ChiRP peaks with our transcripts. We have tried ChiRPing against 4 different transcripts and for all of them we were able to get very good fold enrichments in the genomic locus of each transcript (on average 50 to 200 x over background) capturing native transcription but we cannot find other binding sites in the genome for any of them. We have good reasons to believe that some or all of our transcripts are chromatin localized and we also know that the signal in the genomic loci is RNA dependent because it is dramatically reduced when we ChiRP after knocking down our transcripts of interest.

    Our transcripts are low in expression, on average 3 to 10 copies per cell. Any ideas as to how can we improve/solve the issue? Is it abundance that is limiting the system? Has anyone experienced/solved something similar?
    We tried the normal protocol, and now we are trying with 3 % FA in parallel to glut. Any suggestions would be very welcomed!

    Antonis
    Reply

    Posted by: Antonis A.November 13, 2014, 5:18 AM

    Hi Antonis, I'm afraid the copy number is simply too low. In all of the ChIRP paper published so far I don't think anyone went that low.

    Your enrichment of the genomic loci could be complicated by ChIRP probes pulling down the DNA directly. Do an RNase-control to see if the signal is still there.
    Reply

    Posted by: Ci C.November 13, 2014, 8:52 AM

    Dear Chu,

    Thank you for the prompt reply-yes I know that with such expression levels, ChIRP is challenging. I can try the RNase control but as mentioned I am confident about the RNA-dependence of the signal, because it falls back to LacZ levels when I knock down the transcripts. I guess we will have to try increasing the sensitivity of the experiment. Could an increase in x-linking time help here, have you guys tried 20 min of glut x-linking?
    Reply

    Posted by: Antonis A.November 18, 2014, 3:28 AM

    Hello,
    I enjoyed your paper very much.

    At the end of the video, and in the figure, you mention that in this process you can isolate RNA binding proteins that were associated with the RNA. My questions are: have you had success in doing this? What is the protocol for isolating the proteins? Do you just isolate the protein fraction from the Trizol, or is there a more specialized method? Is there a paper that may lay out use of this method?

    Thank you for your help.
    Reply

    Posted by: jason h.November 26, 2014, 10:15 PM

    Dear ChIRPers, we're happy to announce that you can now officially design and order ChIRP from Biosearch Technologies (Single molecule fish). Scientists developing ChIRP probes using the Stellaris probe designer and following the guidelines from our publication can now email Sheila Semaan at Biosearch Technologies, Inc. for help with ordering the probes in the proper format. Sheila’s contact info is as follows:

    Sheila J. Semaan, Ph.D., Associate Product Manager
    Biosearch Technologies, Inc.
    Email: ssemaan@biosearchtech.com
    Reply

    Posted by: Ci C.December 17, 2014, 9:33 AM

    Dear ChIRP community,

    You can soon study RNA-protein interactions in vivo using ChIRP-mass spec. The story is coming out in Cell on April 2nd. Stay tuned!
    Reply

    Posted by: Ci C.March 25, 2015, 5:42 AM

    Hello, thank you for the protocol! I also want to perform a RNA pulldown using your protocol and i am very excited for your RNA-protein interaction protocol! I have one question regarding the biotin oligos. Do they need to be 3' biotinylated or can they also be 5' biotinylated. Have you tested this? And you say nothing about (salt, HPLC) purification of the oligos. Thanks for answering my questions.
    Reply

    Posted by: Christine C.March 30, 2015, 10:01 AM

    and is it necessary to order Biotin-TEG oligos or is "standard" biotin sufficient?
    Reply

    Posted by: Christine C.March 30, 2015, 11:33 AM

    Hi Christine, thanks for your interest in our technique. Regarding your questions:

    1) 3' biotinylation is preferred, because oligos are synthesized 3' to 5'. This way, we ensure all oligos start with a biotin tag. 5' biotin tags are usually added to a synthesized oligo, and that increases the chance of untagged probes, which is less than ideal. However if you can ensure high tagging efficiency on 5' end, and the cost is advantageous, I don't see why it won't work!

    2) We use a disposable reverse phase column called "Glen-Pak DNA purification cartridge," which is a very efficient and cost-effective system. These are really short DNA probes and HPLC or PAGE would be necessary (also probably can't tell tag from untag probes).

    3) TEG ensures no steric hindrance, I don't have hard evidence on this but I think it's necessary.
    Reply

    Posted by: Ci C.March 30, 2015, 10:03 PM

    Thank you for your answers! As I am performing your protocol with the aim of an RNA-protein pulldown, i have some other questions regarding the protocol, if you don't mind.

    - in the protocol before washed your magnetic beads in unsupplemented lysis buffer and you blocked the magnetic beads using BSA and yeast RNA- in the latest protocol you don't block. Why is that?

    - ChIRP-MS requires 100 Mio cells, ChIRP-seq 20 Mio cells. If i want to perform ChIRP-MS (or western blot) do i need 100 pmol of probe for each 20 Mio cells --> 500 pmol of probe for ChIRP-MS? --> 500 µl beads(??)
    Reply

    Posted by: Christine C.April 8, 2015, 8:12 AM

    Hi Christine,

    - We no longer block as there's no difference in results, and we prefer a more streamlined protocol. BSA would also potentially contaminate ms results.

    - yes scale up beads accordingly. We do 1ml beads per experiment routinely. It gets expensive, but hopefully you'd only have to do it once or twice, and validate with western in a smaller scale later. For western of more abundant chirp hits, I do from 1/5 of cell pellets, but for less abundant ones I still use full amount.

    Reply

    Posted by: Ci C.April 8, 2015, 11:36 PM

    Dear ChIRPers,

    ChIRP mass spec has just come online today at Cell. We're very pleased to introduce a very robust and straight-forward method that's optimized to study lncRNA:protein interactions. Check out the story at:

    http://www.cell.com/cell/abstract/S0092-8674(15)00312-8

    Happy ChIRPing!

    Best,
    Chu
    Reply

    Posted by: Ci C.April 2, 2015, 9:00 PM

    Dear ChIRPers,

    Now you can easily design and order ChIRP probes with the latest optimization from Biosearch Technologies, the company that invented Single Molecule FISH.

    https://www.biosearchtech.com/chirpdesigner/

    Best,
    Chu
    Reply

    Posted by: Ci C.April 2, 2015, 10:24 PM

    Hi, i'm trying to perform ChIRP-western and i have some questions concerning the methods.

    1) for a control, i also want to see if my RNA is present: how can i get a small aliquot of post-ChIRP beads to perform RNA elution with this aliquot
    2) for the protein biotin elution buffer: resuspend beads in what volume?
    3) for ChIRP-MS lysates are incubated with 30µl beads - can i reuse them?

    thank you in advance!
    Reply

    Posted by: Carolin C.April 14, 2015, 7:18 AM

    Hi Carolin,

    thank you for your interested in our method.

    1) yes that's what we we as well. Simply take a 1% or 10% aliquot of your post-wash beads, and extract RNA.

    2) that's dependent on your starting beads volume. But there's a practical constraint, you don't want to have so much volume that after addition of TCA will exceed the capacity of an eppendorf tube.

    3) I'm not sure if I understand your question. Reuse lysate or beads?
    Reply

    Posted by: Ci C.April 15, 2015, 12:20 AM

    thank you for the answers!
    to 2) again: if my beads are 100µl starting volume - so i resuspend 2x in biotin elution buffer and pool. Is 2x100µl then enough? and then add 25% of 100%TCA (50µl)?
    to 3) my question is, if i reuse the beads in the hybridization step. and just add another 70 µl beads (=100µl beads)
    Reply

    Posted by: Carolin C.April 24, 2015, 11:23 AM

    so what do you recommend for my previous questions 2) and 3)

    another question: how do you calculate the amount of RNA retrieved? I retrieve only 10% of my RNA of interest calculated as % of Input. Furthermore,when i took a sample of what hasn't bound (after hybridization and capture of the beads) i get only 15% (fraction of washing doesn't include much RNA). so where has my RNA gone? i repeated several times… degradation?
    Reply

    Posted by: Carolin C.May 20, 2015, 5:13 AM

    Hi, Carolin
    I am also, using ChiRP. Can you tell me the equation you used to calculate the % of your retrieval

    thanks
    Reply

    Posted by: u.June 28, 2016, 1:22 AM

    Dear ChIRPers, we're glad to introduce the Magna ChIRP kit from Millipore. It's a simple pre-made reagent set for scientists who prefer the reliability and user-friendliness of a reputable brand.

    https://www.emdmillipore.com/US/en/product/EZ--Magna-ChIRP-RNA-Interactome-Kit---Isolation-and-characterization-of-non-coding-RNA%3Achromatin-complexes,MM_NF-17-10495

    Happy ChIRPing!

    Best,
    Chu
    Reply

    Posted by: Ci C.April 15, 2015, 12:35 AM

    Hi Chu

    Thanks for sharing this information.
    Did anyone try this kit already and how good is it? Kits are easy to use and save time for reagents.
    Reply

    Posted by: Liu Z.April 16, 2015, 5:17 PM

    Dear Chu,

    Many thanks for your excellent protocol. I'm designing a probe set and have been looking at the probes you designed for HOTAIR and XIST for inspiration. I noticed that some of the probes designed for XIST target the sequence at multiple loci (e.g. Probe 10), albeit ocassionally with a single base mismatch. Either way, I assume this may reduce the number of probes needed. Could I ask whether this was a deliberate design strategy and, if so, does the Biosearchtech ChIRP designer incorporate this strategy?

    Thanks
    Reply

    Posted by: Sebastian V.April 16, 2015, 11:31 AM

    Dear Chu

    I feel exciting when I am reading your chirp paper in molecular cell. I have 2 questions that need your input.

    1) Have you or others you know tried in vivo tissue samples and if so how it works? I realized that your molecular cell and the latest cell paper used cell lines primarily.

    2) If I have a non-coding RNA which is 400 bp alone, and cannot generate more than 8 probes based on the parameters provided by https://www.biosearchtech.com/chirpdesigner/ ? What should I do ? Could I just reduce the spacer length between odd and even probes ? do you think it will increase background due to the increased probe density?

    Thanks

    Zhiyong

    Reply

    Posted by: Liu Z.April 16, 2015, 4:49 PM

    Hi Zhiyong,

    Thank you for your kind words about our paper. Regarding your questions:

    1) Yes we have ChIRPed in drosophila tissues with great success. The key is probably to have small enough chunks of tissue that can be crosslinked effectively and thoroughly.

    2) Email the Biosearchtech team and they can help you manually reduce the number of probes.

    Good luck!

    Best,
    Chu
    Reply

    Posted by: Ci C.May 22, 2015, 11:15 PM

    hey there, i was able to retrieve RNA with your method, but i'm not retrieving RNA bound protein (even though i did see a milky pellet after TCA and acetone treatment). i cross link with UV but the other steps i perform are just the same you are doing.
    i'm not so sure about this competetive elution method by using free d-biotin - and do you no longer add protease inhibitors in your buffer? the beads are simply eluted by using biotin buffer, rotate sample for 20 min,RT and 10 min mixing at 65°C (x2). This is it? then everything should be in my buffer and i continue with TCA precipitation ON.
    sorry if i this sounds stupid to you. i'm just looking for mistakes i could have made...
    Reply

    Posted by: Carolin C.June 10, 2015, 10:32 AM

    Hi Carolin, I'm glad you're able to get RNA yield, that's a very good sign. The reason that you're not getting proteins could be: 1) mass spec instrument not sensitive enough. The quality of your results varies A LOT with who runs your mass spec. We've had amazing experience with Bill Lane at Harvard. 2) UV cross linking is known to be super low yield. Unless your lncRNA is very abundant or you're prepared to start with a huge amount of material, I would suggest formaldehyde as your first step. 3) TCA and cold acetone with give you a tiny pellet no matter what (the detergent acts as a carrier that will precipitate by itself even in the absence of proteins). So unless you see a sizable pellet, the presence of a pellet itself doesn't mean anything. d-biotin elution is complete, assuming you're using the C1 beads and biotin from invitrogen. Protease inhibitors are absent at the elution step, because your solution should be quite pure and free of contaminating proteases at that point assuming good lab practice. Hope this helps!
    Reply

    Posted by: Ci C.June 10, 2015, 5:45 PM

    hey ci! thank you for your answers!
    1) i'm doing western blot as a start, later on i will do mass spec - so thank you for your tip&hint! as i already know a 100% candidate to get retrieved with my (cytoplasmic) mRNA, i stained for this candidate and got no result.
    2) you also mentioned uv-crosslinking in your first protocol, do you have a comment or any experience on the time of cross linking or amount of joules? so far i cross linked 150mj/cm^2 but I'm thinking to increase that. also i need to scale up my cells/beads/probe as you're saying. i do not know if formaldehyde cross linking works in my case, as i have cytosolic mrna.
    3) yes I'm using c1 dynabeads and d-biotin from invitrogen! thanks for the explanation, that helps!

    thank you very much for your quick answers, tips and hints!
    Reply

    Posted by: Carolin C.June 11, 2015, 8:43 AM

    hey there, repeated the experiment - this time i used 200 Mio MEF cells - 10 ml lysate and 1 ml beads, 10µl probe. i also increased cross linking to 300 mj/cm^2 - still no proteins (known RNA binding protein) detectable via western blot. i confirmed RNA pulldown via qPCR after cross linking from a small fraction of beads.
    is sonication necessary?
    any troubleshooting ideas?
    Reply

    Posted by: Carolin C.June 17, 2015, 11:38 AM

    I would start with formaldehyde first. UV is known to cross link very inefficiently, and certain residues of contact may not be amenable to photocrosslinking at all. Yes sonication is necessary to soluble the cell lysate.
    Reply

    Posted by: Ci C.June 19, 2015, 2:37 PM

    i'm using your 25%TCA / Aceton precipitation method to precipitate my proteins. Have you also experienced a pH change (when adding laemmli it turns green/yellow) due to rest TCA? And i think i don't get my pellet resuspended - even after mixing and resuspending. Isn't boiling for 30 min, 95°C destroying the proteins?

    Posted by: Carolin C.July 10, 2015, 7:30 AM

    Yes I have had similar experiences before: pH change usually can be prevented by more careful and thorough acetone washes (although need to care not to disturb pellet), and over drying will cause difficulty in resuspension. I never air dry for over a minute. Boiling is completely fine. Try using the LDS samples buffer / Bis-tris gel system from invitrogen, these work great on chirp samples. If you are still having issues with pH, you can manually adjust a little with a moderate alkaline buffer.

    Posted by: Ci C.July 10, 2015, 10:59 PM

    Hi Carolin,

    I've the same problem as you had. I could only retrieve 10-15% of RNA when compare to input RNA. Could you please share how you sort this problem?

    Thank you in advance!
    Reply

    Posted by: Ganeshkumar A.July 23, 2015, 4:25 AM

    Hi Ganeshkumar, unfortunately i didn't solve the problem… but i found more RNA of interest in my beads when i directly resuspended them in trizol (leaving out proteinase K treatment) - since i'm interested in my proteins bound to the RNA the proteinase K treatment step is of no importance to me. But so far i still couldn't retrieve my RNA bound proteins.
    Reply

    Posted by: Carolin C.July 23, 2015, 5:19 AM

    Hi Carolin,

    Thanks for your immediate reply. I'll also avoid Proteinase K step and try again.
    I've one more question. We always do two step RT-qPCR. So, the amount of RNA that I retrieve isn't enough to perform a RT reaction. How do you do your RT-qPCR? This question maybe a bit stupid, but i really wanna sort out all the issues as early as possible.

    Thanks in advance.
    Reply

    Posted by: Ganeshkumar A.July 23, 2015, 5:27 AM

    Hello,
    my RNA amount is also quite low, measured by Nanodrop (Once i measured with qubit and could't get anything). I'm using max. volume possible for RT reaction and adapt for input, not bound… Then i perform qPCR with my cDNA

    But i suppose you cannot skip proteinase K step when your lysate is formaldehyde treated, or you need to boil it first to reverse the crosslink. i diluted my beads in trizol when i had non-crosslinked samples to check for retrieval percentage

    Posted by: Carolin C.July 23, 2015, 7:12 AM

    We always do a one-step qRT-PCR (i.e. each qPCR well runs it's own RT). This in my hands is much more sensitive and convenient as well (you don't have to measure RNA or cDNA, just load equal portions of each sample). We use Strategene Brilliant II SYBR reagents and Roche 480 machine. Let me know if this helps with yield detected.

    Posted by: Ci C.August 4, 2015, 8:32 AM

    Hi Ci,

    I did gene specific cDNA synthesis with 10ng of RNA from all conditions and treated with RNase H and A (in excess) and then proceeded to qPCR. This worked good for me and I'm getting around 14-25 fold enrichment in my odd and even probe samples when compared to input.

    Thank you and Carolin for your suggestions.

    Posted by: Ganeshkumar A.August 4, 2015, 10:43 AM

    Hello,
    i have not yet retrieved any RNA bound protein, so i'm thinking about what issues to sort out. i have to say i can retrieve my RNA of interest (but only max. 20% of input)

    i'm wondering about the protein amount in your formaldehyde cross linked, sonicated and hypotone lysed cell lysats. I experienced the protein amount (measured with Bradford) to be 5-8 fold lower compared to uncross linked or Uv cross linked cells treated with a NP40 lysis buffer.
    Why is that, and will it affect my experiments? Any ideas why the protein amount is so low?
    Thank you in advance!
    Reply

    Posted by: Christine C.July 23, 2015, 5:47 AM

    Hi Christine, off the top of my head the two main reason for less protein detected upon cross linking is 1) insufficient sonication, and 2) insufficient reverse cross linking (i.e. boiling). Regarding 1), you have to titrate sonication time of your lysate, and pick the minimum sonication required to release all proteins and RNA of interest. To test this, precipitate the lysate post sonication for 10 min at max speed, and run qRTPCR against your RNA of interest and western against the protein on both the supernatant and pellet, the latter resuspended in appropriate buffer. This way you'll know if there's anything left to be solubilized. 3% cross linked lysates can take significantly longer to solublized than your typical ChIP cross linking.

    Regarding 2), do no less than 30min boiling in sample buffer. I've titrate that also, and 10min or 20min recovers much less protein.

    Let me know if these help and let's start from here. Good luck!
    Reply

    Posted by: Ci C.August 4, 2015, 8:29 AM

    Hello,
    Many thanks again for this superb protocol. I'm currently trying to optimise this for mRNA pull-down from fixed cells. I've tried various fixing conditions in parallel and dot-blotted for my RBP of interest vs a non-binding protein (GAPDH) after performing ChIRP. I found that 1% glutaraldehyde (10 min) samples blotted strongly for my RBP, but produced an equally strong signal for GAPDH. The latter was also the case for 3% formaldehyde (10 min), but blots of both GAPDH and my RBP weren't as strong. The only sample that showed a slightly stronger signal of the RBP vs GAPDH was 1% formaldehyde (10 min), but both signals were quite weak. All samples were sonicated for several 30 sec ON and 45 OFF cycles at maximum intensity before foaming to produce sheared RNA of <500 bp in optically clear samples.

    I tested the expression of mRNA of interest in samples pulled down with my tiling oligo set compared with a run using non-targeting scrabled oligos. A capture on unfixed cells produced a very high target yield in the tiling oligo sample and no mRNA in the scrambled control sample. However, performing the same pull down in the 1% formaldehyde-fixed+sonicated samples dramatically increased background (GAPDH mRNA) compared to the scrambled control. I'm curious if you found anything similar during the development of ChIRP and ChIRP-MS and if you might have any solutions? Would extra sonication perhaps help?

    Many thanks

    Sebastian Vencken PhD
    Post-Doctoral Researcher
    Reply

    Posted by: Sebastian V.September 13, 2015, 8:06 PM

    Hi Sebastian,

    Have you had any luck adapting this technique to mRNA capture?

    Best,
    Mike
    Reply

    Posted by: Michael K.December 7, 2016, 4:48 PM

    Mixed results to be honest. We went forward with 1% formaldehyde and tried different forms of sonication (incl. covaris which didn't give us effective fragmentation). Biased background was still an issue and the copurification of our RBP (Ago+miRNA) was much lower than expected. This has apparently successfully been performed before (https://www.ncbi.nlm.nih.gov/pubmed/23325846) but we didn't manage to reproduce these results for our target.
    Reply

    Posted by: Sebastian V.December 8, 2016, 4:15 AM

    Thanks for the feedback. Does your miR-CATCH work well/better? Similar idea, no sonication, and probes attached to beads first. Have you ever tried to use this to look for bound proteins?

    I also ran across this mRNA pulldown from yeast (https://www.ncbi.nlm.nih.gov/pubmed/27641505). They also attached the oligos to the beads first, but the buffers look more like ChIRP.

    Reply

    Posted by: Michael K.December 8, 2016, 11:44 AM

    We've had some pretty good results (and publications) from miR-CATCH, but have also discovered that the performance can be very dependent on both the target and oligo design.
    With thorough optimisation we managed to reduce background in miR-CATCH, but each target was a new challenge in this respect.

    We hoped that ChIRP would significantly reduce the issue of background, but we have found balancing fragmentation and signal to be difficult. Dot blots for Ago2 gave us very high background signal in scrambled controls when using gluteraldehyde, while 1% formaldehyde gave some better signal differentiation, but at reduced strength. If I'd take ChIRP further for mRNA:RBP(:miRNA) capture, I'd look at the sonication and fixing steps again as there may be further room for improvement. Unfortunately, I've moved to other things for now.
    Reply

    Posted by: Sebastian V.December 9, 2016, 10:42 AM

    Hello!

    Many thanks for the detailed protocol, however i am having trouble at crosslinking and sonication steps! I am sorry if it sounds silly but I just want to identifiy what I am missing/doing wrong.

    In the near future, I want to scale up for ChIRP MS, however first want to get these steps right as CHIRP MS would consume 10 times the beads.

    Crosslinking - When I add Glycine to quench formaldehyde, it doesnt change color?

    Sonication - As used by your team, I am using Bioruptor to sonicate the Hela S3 lysate (20 Mi. cells/1ml supplemeted lysis buffer) in 15 ml falcon (Corning) with a probe attached to the cap. I cant seem to get a clear lysate as shown in your video. I have tried sonicating starting from 30 cycles to 300 cycles and it just froths towards the end. After centrifugation to remove cell debris, the lysate is still considerably cloudy.

    I see that a lot of people are working on optimising this method and would appreciate any tips / tricks that might help me get going.

    Looking forward to some suggestions.

    Best regards,
    Minakshi
    PhD Student

    Reply

    Posted by: Minakshi G.September 30, 2015, 5:06 AM

    Hi Minakshi, thanks for your interest in our protocol. Re your questions:

    - glycine doesn't change the color of formaldehyde, just glutaraldehyde.
    - check our bioruptor to make sure power is normal and probes are centered correctly. It shouldn't take more than a couple hours even for hardy cell lines. Frosting is strange and indicates to me that you may not be centering the probe correctly and thus losing efficiency.
    Reply

    Posted by: Ci C.November 2, 2015, 2:46 PM

    Hi,
    I would have a question regarding the usage of formamide. My concern is that once the bottle is opened and the
    formamide is exposed to oxygen, it will begin to oxidize to formic acid. Do you take any precautions to prevent oxidation? How do you store formamide for further use? Sigma suggests to purge formamide with nitrogen and store it frozen to prevent oxidation.

    Thanks a lot for your help.

    Best wishes,
    matjaz
    Reply

    Posted by: Matjaz B.November 2, 2015, 4:53 AM

    Hi Matjaz, thank you for your interest in our technique. formamide is indeed subject to oxidation and ionization. Re-deionization is laborious, so we tried our best to protect the reagents by parafilming around the bottle cap after each use and store it at 4C. I usually run through a 1 liter bottle in a year with repeated opening and closing, and have not observed any effect on results over time. Hope this helps.
    Reply

    Posted by: Ci C.November 2, 2015, 2:44 PM

    Hello,

    I have another technical question. Even though our probes are very specific (we detect target RNA using different probes with high specificity), we have problem with high DNA background signal. Have you also had a similar problem? One way to resolve the issue would be to pre-clear lysates with beads before the hybridisation step. Have you ever implemented this step? If yes, have you done the pre-clearing in lysis buffer with or without hybridisation buffer?

    Many thanks,

    - matjaz
    Reply

    Posted by: Matjaz B.November 3, 2015, 7:36 AM

    Hi Ci,

    Thanks for providing such an excellent protocol. I have a question about probe design. The 3' half of lncRNA I interested in (5kb in length) was fully cover by repeat elements. Because ChIRP probe Designer ignored the repeat sequence when designing probe, I found almost no probe target to the 3' half of this lncRNA. I'm afraid that I will miss all proteins bind to the 3' end of lncRNA if I only use the probe target to the 5' end of lncRNA. I wonder how do you handle lncRNA contains large number of repeat sequence?

    Thanks for any help.

    Sincerely,
    Jian-You
    Reply

    Posted by: Jian-You L.December 3, 2015, 9:48 PM

    Hi all!

    Thank you for sharing this protocol!
    I have just analysed the RT-qPCR results coming from the RNA samples in order to analyse the lncRNA retrieval and I have retrieved a very small percentage of the lncRNA. From the input and the ChIRP samples I have used the same volume of sample (for the qPCR, around 36ng for the input and 21g for the ChIRP) and I have obtained a Ct of 30 for the input and 37 for the ChIRP measuring my lncRNA of interest. How do you normalise the results? Do you use the same volume or ng of the different RNA samples? Do you use a normalisation method similar to the ChIP one? https://www.thermofisher.com/uk/en/home/life-science/epigenetics-noncoding-rna-research/chromatin-remodeling/chromatin-immunoprecipitation-chip/chip-analysis.html

    Thank you in advance

    Isabel
    Reply

    Posted by: Isabel R.December 17, 2015, 9:30 AM

    Hi, Isabel
    we experience along time to optimize the protocol in our lab around 9 month for lncRNA and we found the result unstable for different lncRNAs but for your normalization we usually use a normalized methods like Chip experiment for both GAPDH and our target,
    Hope that helps
    for more contact amr@mail.ustc.edu.cn
    Reply

    Posted by: u.December 8, 2016, 7:17 PM

    Dear Chu:
    Recently I'm trying to perform ChIRP experiments and your detailed protocol is very helpful. However, I have some questions about cell lysis and sonication.
    1. I find that the supplemented lysis buffer cannot lyse the crosslinked cells. The crosslinked cells are only resuspended, after a few minutes they will sink to the bottom of the tube, is it normal?
    2. You emphasize that lysis buffer should be added fresh Protease Inhibitor, PMSF and Superase-in then proceed immediately to sonication after resuspended smooth, however, a sample will be added appropriate supplemented lysis buffer in proportion then devided into several parts to sonicate by limiting ultrasound equipment, and will it be affected?
    3. I only have a Covaris available for sonication in lab, I tried many times but could not find an appropriate setting. My DNA size distribution is normally a bright band of gDNA at the top, a bright band above 2000bp and a diffuse band from 2000bp to 100bp, if I continued to sonicate, the diffuse band would below 100bp, and there were considerably cloudy after centrifuged the sonicated samples. I want to know what and how much influence if DNA smear is less than 100bp or more than 500 bp? Do you have any advice regarding it?
    Looking forward to some suggestions, thank you in advance!
    Best regards
    mixue
    Ph.D. Student
    Reply

    Posted by: he j.July 17, 2016, 3:37 AM

    Dear Dr CHU.
    First of all, sorry for my english, but I'm french.
    I work on lncRNA and I really appreciate your two articles on ChIRP and Chirp-MS, and since some months I try to set it up, but unfortunately without result until now.
    => I designed the probes as you know but my lncRNA is about 2.3kb so i was able to design only 26 probes. I also ordered your Positive and Negative Control.
    => I amplified the cells until 200 to 300 million and I cross linked them with 3% formaldehyde 30min and stop it with Glycine 0.125M 5 to 10 min, as you mention.
    => My sonication was tested in order to have a smear of DNA between 100 and 600bp and it took between 120 and 140min. I use a Bioruptore in cold room and I have to change water and ice every 15min in order to maintain the low temperature. Samples are centrifuged and debris are removed.
    => I use 2 to 3 aliquots of 1mL of lysed cells for each test, and the indicated quantity of probes. Of course before incubation, samples are pre-incubated with prewashed beads.
    => First incubation is done at 37 ° C in a shaking system, overnight. Second incubation (after addition of the prewashed beads) is done at 37 ° C for 1 hour.
    => Wash is done 5 times with wash buffer, then samples are eluted (using elution buffer freshly prepared with Sigma-Aldrich Biotine) and precipitated with TCA and Aceton. Pellet are directly dissolved in Laemmli, incubated 30min at 95°C and loaded with 12% acrylamide gel. After 1h at 200V the gel is stained with Coomassie Blue and destained 3 times 45min.
    I found NO BANDS even with Positive and negative controls, but after elution, when I checked the presence of my lncRNA in the sample selected with my probes, the result is positive.

    Could you tell me what are the possible mistakes that I could do ....?
    Thanks a lot.
    Reply

    Posted by: Sebastien C.January 3, 2017, 5:02 AM

    Dear Dr. Chu,

    Good day! I am Anchilie, a PhD student, and I am really interested in applying your discovered technique in plants. I am currently doing the ChiRP-MS in Arabidopsis but I don't seem to get any RNA-bound proteins in my sample.

    I would just like to briefly tell you how I do the ChiRP-MS in plants. First, I harvest 3g of 14-day old seedlings and crosslinked it with 1% formaldehyde. From these, I then do nuclear extraction and do sonication. From the 1mL sonicated chromatin, I dilute it with 2mL of hybridization buffer, add 1ul probe and 100ul of beads and incubated then in 37C for O/N or 4 hours at 37C. From then I wash the beads 5x and precipitated the proteins using the biotin elution buffer and TCA method. The samples in 1x Laemmli buffer were submitted to LC/MS. I also check the enrichment in the qPCR and are enriched by 500-fold. However, upon analyzing my samples, the mass spec results only detected mitochondrial and chloroplastic proteins in the control and non-coding RNA

    I also tried many things like using 3% formaldehyde and UV crosslinking. I also did direct boiling of beads in Laemmli buffer. I also tried increasing the amount of material 10x accompanied with increasing the probe and the beads.
    But it doesn't really help.

    I would really appreciate to receive some feedback from you and your group with this matter.

    Best regards,
    Anchilie
    Reply

    Posted by: Anchilie M.February 27, 2017, 4:49 AM

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