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.

1. Probe Design
Design anti-sense DNA tiling probes for selective retrieval of RNA target by ChIRP.
<ol>
	<li>Design anti-sense oligo probes using the online probe designer at <a href="http://singlemoleculefish.com">singlemoleculefish.com</a> 18.</li>
	<li>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.</li>
	<li>Order anti-sense DNA probes with BiotinTEG at 3-prime end.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Harvest Cells
Collect cells that will be used for ChIRP experiment.
<ol>
	<li>Grow cells in tissue culture plates or flasks to confluency. Rinse with phosphate buffered saline (PBS) once and trypsinize. Quench trypsin with &gt;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.</li>
	<li>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.</li>
</ol>
3. Cross-link Cells and Collect Cell Pellet
Crosslink collected cells with glutaraldehyde to preserve RNA-Chromatin interactions and prepare cell pellet.
<ol>
	<li>Perform all steps at room temperature.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Quench the cross-linking reaction with 1/10th volume of 1.25 M glycine at room temperature for 5 min.</li>
	<li>Spin at 2000RCF for 5 min. Aspirate supernatant and wash pellet with 20 mL chilled PBS once, spinning at 2000RCF for 5 min.</li>
	<li>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.</li>
	<li>Flash-freeze the cell pellets in liquid nitrogen and store at -80 °C indefinitely.</li>
</ol>
4. Cell Lysis
Lyse crosslinked cells to prepare cell lysate.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Add 10X volume supplemented Lysis Buffer to each tube and resuspend the pellet. For small pellets &lt;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.</li>
</ol>
5. Sonication
Shear DNA by sonicating crosslinked cell lysates.
<ol>
	<li>Sonicate cell lysate in Bioruptor in 15 ml Falcon tubes. Use &lt;1.5 ml lysate in each tube, and for faster sonication, sonicate no more than two tubes at a time.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
6. ChIRP
Hybridize biotinylated DNA probes to RNA and isolate bound chromatin.
<ol>
	<li>Thaw tubes of chromatin at room temperature.</li>
	<li>Prepare Hybridization Buffer (see buffer list, prepare 2 ml per ml of chromatin). Vortex to mix.</li>
	<li>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.</li>
	<li>Transfer 1 mL chromatin to 15 mL Falcon tube. Add 2 mL Hybridization Buffer to each tube. For total volume &lt;1.5 ml, use Eppendorf tubes.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Prepare Wash Buffer (5 mL per sample). Vortex to mix. Pre-warm to 37 °C. Add PMSF before use.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
7. RNA Isolation
Extract RNA fraction from ChIRP samples to quantitate by qRT-PCR.
<ol>
	<li>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.</li>
	<li>Briefly spin down all tubes and boil samples for 10 min on heat block at 95 °C.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Remove ~400 μL aqueous supernatant, avoiding organic and interface.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Use 1 μL RNA isolate per well for qRT-PCR analysis to confirm lncRNA retrieval. GAPDH is often used as a negative control.</li>
</ol>
8. DNA Isolation
Extract DNA fraction from ChIRP samples to identify by sequencing or quantitate by qPCR.
<ol>
	<li>Prepare DNA Elution Buffer (see buffer list), 150 μl per sample, including DNA INPUT.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Separate beads and supernatant on DynaMag-2 magnetic strip. Remove supernatant and add to labeled tubes.</li>
	<li>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).</li>
	<li>Add 15 μL PK to each sample. Incubate at 50 °C for 45 min with shaking.</li>
	<li>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.</li>
	<li>Spin samples at 16100RCF for 30 min at 4 °C.</li>
	<li>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.</li>
	<li>DNA samples are ready for analysis by qPCR or preparation of high-throughput sequencing libraries per Illumina protocol.</li>
</ol>
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.
<img alt="ChIRP diagram: Crosslink, sonicate, hybridize RNA; purify on beads; RNA-protein interaction analysis." src="/files/ftp_upload/3912/3912fig1.jpg" data-altupdatedat="1747911167000" data-alt="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
<img alt="Bar graph of RNA retrieval, ChIRP analysis; comparing TERC vs GAPDH, showing experimental outcome." src="/files/ftp_upload/3912/3912fig2.jpg" data-altupdatedat="1747911167000" data-alt="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
<img alt="HOTAIR qPCR results in bar graph, genes analysis, ChIP-RNA input percentage comparison." src="/files/ftp_upload/3912/3912fig3.jpg" data-altupdatedat="1747911167000" data-alt="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
<img alt="Chromatin interaction CHIRP-seq graph; Refseq genes; roX2 binding on chrX." src="/files/ftp_upload/3912/3912fig4.jpg" data-altupdatedat="1747911167000" data-alt="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

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

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.

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

chromatin isolation by rna purification (chirp)

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.

Watch this Scientific Journal Video about Chromatin Isolation by RNA Purification ChIRP at JoVE.com

Chromatin Isolation by RNA Purification ChIRP

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.

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

chromatin-isolation-by-rna-purification-chirp

Research

JoVE Journal

Biology

86.3K Views.  Stanford University School of Medicine. 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.

Video: Chromatin Isolation by RNA Purification ChIRP

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

Genetics

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

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

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

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

Biochemistry

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Chromatin Extraction from Frozen Chimeric Liver Tissue for Chromatin Immunoprecipitation Analysis

Crosslinking Chromatin Immunoprecipitation (X-ChIP) is a widely used technique to assess levels of histone marks and occupancy of transcription factors on host and/or pathogen chromatin. Chromatin preparation from tissues creates additional challenges that need to be overcome to obtain reproducible and reliable protocols comparable to those used for cell culture. Tissue disruption and fixation are critical steps to achieve efficient shearing of chromatin. Coexistence of different cell types and clusters may also require different shearing times to reach optimal fragment size and hinders shearing reproducibility. The purpose of this method is to achieve reliable and reproducible host chromatin preparations from frozen tissue (liver) suitable for both ChIP-qPCR and next generation sequencing (NGS) applications. We observed that the combination of liquid nitrogen tissue pulverization followed by homogenization leads to increased reproducibility compared to homogenization only, since it provides a suspension consisting mostly of dissociated single cells that can be efficiently sheared. Moreover, the fixation step should be performed under mild rotation to provide homogeneous crosslinking. The fixed material is then suitable for buffer-based nuclei isolation, to reduce contamination of cytoplasmic protein and pathogen DNAs and RNAs (when applicable), avoiding time-consuming centrifugation gradients. Subsequent sonication will complete nuclear lysis and shear the chromatin, producing a specific size range according to the chosen shearing conditions. The size range should fall between 100 and 300 nt for NGS applications, while it could be higher (300-700 nt) for ChIP-qPCR analysis. Such protocol adaptations can greatly improve chromatin analyses from frozen tissue specimens.

This protocol focuses on chromatin preparation from snap frozen tissues and it is suitable for Crosslinking Chromatin Immunoprecipitation (X-ChIP) followed by either quantitative PCR analysis (X-ChIP-qPCR) or next generation sequencing approaches (X-ChIP-seq).

Crosslinking Chromatin Immunoprecipitation (X-ChIP) is a widely used technique to assess levels of histone marks and occupancy of transcription factors on ...