This work was supported by Wayne State University, Karmanos Cancer Institute, a grant from the National Heart, Lung and Blood Institute (1K22HL126842-01A1) awarded to B.L.K. This work utilized the Wayne State University High Performance Computing Grid for computational resources (&#60;https://www.grid.wayne.edu/&#62;). We thank Jiji Kurup for assistance with CARIP-Seq data analysis.
AUTHORS&#8217; CONTRIBUTIONS:
B.L.K. conceived of the CARIP-Seq method, designed and carried out the ChIP-Seq and CARIP-Seq experiments, analyzed the sequencing data, and drafted the manuscript. All authors have read and approved the final version of this manuscript.

1. Culture of Mouse ES Cells in Feeder-Free Conditions.
NOTE: Mouse ES cells are conventionally cultured in media on a cell culture dish coated with gelatin and a mono-layer of mouse embryonic fibroblasts (MEF), which have been mitotically inactivated (iMEFs). However, MEFs should be removed prior to downstream epigenetic or expression analyses to prevent the contamination of MEF-associated chromatin and RNA.
<ol>
	<li>Preparation of a feeder layer: Gelatin coat a 6-well cell culture plate b...

<ol>
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The authors declare no conflict of interest.

ChIP-Seq is a useful method to evaluate the location of global protein-DNA interactions (e.g., transcription factors/histone modifying enzymes/histone modifications and DNA) in ES cells, while the newly developed CARIP-Seq protocol is useful in interrogating genome-wide association of RNAs with chromatin constituents. ChIP-Seq is a fundamental tool that is used to evaluate epigenetic landscapes of ES cells and other cell types. The quality of ChIP-Seq and CARIP-Seq libraries is largely dependent on ChIP-grade an...

Embryonic stem (ES) cell fate decisions are regulated by communication between extracellular signals and a host of transcriptional-regulators, including histone modifiers, and post-translation modification of histone tails. These interactions facilitate chromatin accessibility and packaging of chromatin into one of two states: euchromatin, which is open and transcriptionally active, and heterochromatin, which is compact and generally transcriptionally inactive. Transcription factors with DNA sequence-specific binding affinities and epigenetic modifiers associate with euchromatic regions to participate in controlling gene expression. Next-generation sequencing methods, including ChIP-Seq1, have been instrumental in mapping genome-wide transcriptional networks that are fundamental for ES cell self-renewal and pluripotency2,3,4,5,6. Moreover, while RNA immunopreciation followed by next-generation sequencing (RIP-Seq)7 evaluations of RNA-protein interactions suggest that DNA binding proteins interact with RNAs to regulate transcriptional events7,8,9,10,11,12, few studies have investigated the genome-wide localization of RNAs associated with chromatin12, or global interactions between RNA and histone modifications. Long non-coding RNAs (lncRNAs) are one class of RNAs which have been found to regulate the activity of chromatin-associated proteins13,14,15. For example, Xist is a lncRNA that regulates, in female mammalian cells, inactivation of one X chromosome, through the recruitment of epigenetic repressors16,17. However, the full spectrum of RNAs associated with chromatin is largely unknown. Here, we describe a novel protocol, chromatin-associated RNA immunoprecipitation (CARIP) followed by next-generation sequencing (CARIP-Seq), to identify chromatin-associated RNAs on a genome-wide basis in ES cells, including library preparation for next-generation sequencing, and ChIP-Seq to map global occupancy of histone modifications, transcription factors, and epigenetic modifiers. Unlike other RIP-Seq methods7, CARIP-Seq includes crosslinking and sonication steps, which allow for the direct identification of RNAs associated with chromatin. Together, ChIP-Seq is a powerful tool to identify genome-wide protein-DNA interactions, while CARIP-Seq is a powerful method to survey RNAs associated with chromatin components.

<table>
	<tbody>
		<tr>
			<td>Mouse leukemia inhibitory factor (ESGRO LIF)</td>
			<td>EMD Millipore</td>
			<td></td>
			<td>106 or 107 units</td>
		</tr>
		<tr>
			<td>GSK3&#946; inhibitor CHIR99021 (GSK3i)</td>
			<td>Stemgent</td>
			<td></td>
			<td>Solvent: DMSO 10mM</td>
		</tr>
		<tr>
			<td>MEK inhibitor PD0325901 (MEKi)</td>
			<td>Stemgent</td>
			<td></td>
			<td>Solvent: DMSO 10mM</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM), high glucose</td>
			<td>Invitrogen</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (phosphate buffered saline)</td>
			<td>Invitrogen</td>
			<td>10010031</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (50 mM)</td>
			<td>Gibco</td>
			<td>31350010</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Gibco</td>
			<td>11140076</td>
			<td></td>
		</tr>
		<tr>
			<td>EmbryoMax ES Cell Qualified Fetal Bovine Serum, 500 ml</td>
			<td>EMD Millipore</td>
			<td>ES-009-B</td>
			<td></td>
		</tr>
		<tr>
			<td>EmbryoMax 0.1% Gelatin Solution</td>
			<td>EMD Millipore</td>
			<td>ES-006-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>G7126-500G</td>
			<td>1.25 M stock conentration in water</td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma</td>
			<td>F8775-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TE (Tris EDTA) pH 8.0 1X</td>
			<td>Quality Biological</td>
			<td>351-011-131</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF) solution</td>
			<td>Sigma</td>
			<td>93482-250ML-F</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11697498001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate solution (20%)</td>
			<td>Quality Biological</td>
			<td>A611-0837-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Q125 sonifier</td>
			<td>Qsonica</td>
			<td>4422</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100 solution</td>
			<td>Sigma</td>
			<td>93443-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma</td>
			<td>30970</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Protein G for Immunoprecipitation</td>
			<td>Invitrogen</td>
			<td>10004D</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Protein A for Immunoprecipitation</td>
			<td>Invitrogen</td>
			<td>10002D</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Protein A/Protein G and Magnet Starter Pack</td>
			<td>Invitrogen</td>
			<td>10015D</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium chloride</td>
			<td>Sigma</td>
			<td>L4408</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL CA-630</td>
			<td>Sigma</td>
			<td>I8896</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads mRNA Purification Kit (for mRNA purification from total RNA preps)</td>
			<td>Invitrogen</td>
			<td>61006</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript Double-Stranded cDNA Synthesis Kit</td>
			<td>Invitrogen</td>
			<td>11917010</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>End-It DNA End-Repair Kit</td>
			<td>Lucigen</td>
			<td>ER81050</td>
			<td></td>
		</tr>
		<tr>
			<td>Klenow Fragment (3&#39;&#8594;5&#39; exo-)</td>
			<td>NEB</td>
			<td>M0212L</td>
			<td></td>
		</tr>
		<tr>
			<td>dATP (10 mM)</td>
			<td>Invitrogen</td>
			<td>18252015</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>NEB</td>
			<td>M0202L</td>
			<td></td>
		</tr>
		<tr>
			<td>TrackIt 1 Kb Plus DNA Ladder</td>
			<td>Invitrogen</td>
			<td>10488085</td>
			<td></td>
		</tr>
		<tr>
			<td>E-gel EX agarose gels, 2%</td>
			<td>Invitrogen</td>
			<td>G402002</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion high-fidelity 2X master mix with HF buffer</td>
			<td>Thermo Fisher</td>
			<td>F531LPM</td>
			<td></td>
		</tr>
		<tr>
			<td>ChIPAb+ Trimethyl-Histone H3 (Lys4) - ChIP Validated Antibody</td>
			<td>EMD Millipore</td>
			<td>17-614</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)</td>
			<td>Abcam</td>
			<td>ab32356</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Flat Bottom Cell Culture Plates (6-well)</td>
			<td>Fisher</td>
			<td>720083</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Standard Tissue Culture Dishes (10cm)</td>
			<td>Fisher</td>
			<td>08772E</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioruptor pico</td>
			<td>Diagenode</td>
			<td>B01010001</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Flurometer 2.0</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Thermo Fisher</td>
			<td>Q32851</td>
			<td></td>
		</tr>
		<tr>
			<td>MinElute Reaction Cleanup Kit</td>
			<td>Qiagen</td>
			<td>28204</td>
			<td></td>
		</tr>
		<tr>
			<td>MinElute Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28604</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Histone H4 (tri methyl K20) antibody - ChIP Grade (ab9053)</td>
			<td>Abcam</td>
			<td>ab9053</td>
			<td></td>
		</tr>
		<tr>
			<td>Labquake rotator</td>
			<td>Thermo Fisher</td>
			<td>400110Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumina HiSeq platform</td>
			<td>Illumina</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TURBO DNase</td>
			<td>Invitrogen</td>
			<td>AM2238</td>
			<td></td>
		</tr>
	</tbody>
</table>

carip-seq and chip-seq: methods to identify chromatin-associated rnas and protein-dna interactions in embryonic stem cells

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, epigenetic regulators, and post-translation modifications of histones that combinatorially influence the gene expression state of nearby genes. RNA has also been shown to interact with various proteins to regulate chromatin dynamics&#160;and gene expression. Chromatin-associated RNA immunoprecipitation (CARIP) followed by next-generation sequencing (CARIP-Seq) is a novel method to survey RNAs associated with chromatin proteins, while chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq) is a powerful genomics technique to map the location of post-translational modification of histones, transcription factors, and epigenetic modifiers on a global-scale in ES cells. Here, we describe methods to perform CARIP-Seq and ChIP-Seq, including library construction for next-generation sequencing, to generate global chromatin-associated RNA and epigenomic maps in ES cells.

We successfully interrogated the genome-wide binding of H3K4me3, H3K4me2, and KDM5B in ES cells using this ChIP-Seq protocol6. ES cells were cultured in feeder-free conditions (Figure 1), and cross-linked as described above. Sonication was subsequently performed as described in step 3.2 of the ChIP-Seq protocol, and evaluated by running DNA on a 2% agarose gel (Figure 2). Next, ChIP was performed as descri...

Watch this Scientific Journal Video about CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells at JoVE.com

CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells

Here, we describe methods to perform ChIP-Seq and CARIP-Seq, including library preparation for next-generation sequencing, to generate global epigenomic and chromatin-associated RNA maps in ES cells.

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, ...

The overall goal of CARIP-Seq is to survey genome-wide chromatin-associated RNAs. The goal of ChIP-Seq is to evaluate protein-DNA interactions on a global scale in embryonic stem cells. These methods can help answer key questions in the epigenetics and stem cell fields, identify the genome location transcription factors or epigenetic regulator binding or histone modification occupancy or chromatin-associated RNAs in embryonic stem cells.The main advantage of these techniques is that they allow for the genome-wide unbiased interrogation of RNAs associated with chromatin constituents and global identification of protein DNA interactions. Though these methods can provide insight into embryonic stem cell biology, they can also be applied to other mammalian cell types to study cancer biology or other human diseases. To harvest the embryonic stem cells, wash the cells first with phosphate buffered saline.Then, detach the cells with 0.25%trypsin, pre-warmed to 37 degrees Celsius. After trypsinizing, resuspend the cells in pre-warmed MEF medium, supplemented with 10%fetal bovine serum. Then, add 37%formaldehyde to the cells in a conical tube to a final concentration of 1%and incubate at 37 degrees Celsius for eight minutes to cross-link the cells.To stop the fixation reaction, add 1.25 molar glycine pre-chilled at four degrees Celsius to a final concentration of 0.125 molar. Then, place the tube on a rotator and invert it at eight revolutions per minute for five minutes at room temperature. After five minutes, centrifuge the sample at 300g for five minutes at room temperature.After the centrifugation is over, discard the supernatant. Then, wash the pellet with pre-chilled phosphate buffered saline and again centrifuge the sample. Then, remove the phosphate buffered saline and freeze the cells in liquid nitrogen for a minute.Once the embryonic stem cells are thawed on ice, resuspend the pellet in 2.5 milliliters of sonication buffer. To study transcription factors or epigenetic factors, resuspend the cell pellet in sonication buffer without sodium dodecyl sulfate. For histone modifications, resuspend the cell pellet in buffer containing sodium dodecyl sulfate.Next, for chromatin immunoprecipitation, sonicate the chromatin for 14 to 18 cycles at 40%amplitude with 30 seconds on and 30 seconds rest mode. To solubilize the chromatin for the protein factor ChIP, supplement the sonication buffer with 28 microliters of 10%sodium dodecyl sulfate. For protein factors in histone modifications, add 28 microliters of sodium deoxycholate and 0.28 milliliters of 10%Triton X-100 and mix well.Then, centrifuge the sample at 10, 000g for 10 minutes at four degrees Celsius to pre-clear the chromatin. In a 1.5 milliliter low binding tube, add 40 microliters of A or G magnetic beads. Then, wash the beads with 600 microliters of phosphate buffered saline and transfer the tube with the beads on a rack containing a magnet and incubate for a minute at room temperature.Then, remove the phosphate buffered saline and again resuspend the beads in 100 microliters of phosphate buffered saline. To the tube, add two to four micrograms of antibody and incubate it by placing on a rotator for 40 minutes at eight revolutions per minute to promote antibody binding to the beads. Then, add 200 microliters of phosphate buffered saline to wash the beads.After washing, incubate the sonicated chromatin extract on a rotator at four degrees Celsius overnight to immunoprecipitate the chromatin-bound proteins. The next day, wash the beads and incubate with RIPA buffer, RIPA buffer with salt, lithium chloride buffer, Tris-EDTA buffer with Triton X-100, and Tris-EDTA buffer, each for 10 minutes on a rotator. To decrosslink for ChIP, resuspend the beads in 100 microliters of Tris-EDTA buffer.Then add three microliters of 10%sodium dodecyl sulfate and five microliters of 20 milligrams per milliliter of Proteinase K, and incubate the beads at 65 degrees Celsius overnight. After incubation, the next day, invert the tubes several times, leave them on the magnetic rack, and transfer the supernatant to a fresh tube. Then, wash the magnetic beads by adding 100 microliters of Tris-EDTA buffer, containing 0.5 molar sodium chloride and combine the two supernatants.To decrosslink for CARIP, resuspend the beads in 100 microliters of Tris-EDTA buffer. Then, add three microliters of 10%sodium dodecyl sulfate and five microliters of 20 milligrams per milliliter of Proteinase K and incubate the beads at 65 degrees Celsius for four to 12 hours. Again, invert the tube several times and place on the magnetic rack to transfer the supernatant to a fresh tube.Then, wash the magnetic beads by adding 100 microliters of Tris-EDTA buffer, containing 0.5 molar sodium chloride and then combine the two supernatants. Finally, isolate the RNA sample by using phenol chloroform isoamyl alcohol in 25 to 24 to one volume by volume ratio. Next, use DNase to remove DNA from the chromatin associated immunoprecipitated RNA sample.Then, add 10X DNase buffer to the CARIP sample and adjust the final volume to 1X concentration. Then, incubate the sample at 37 degrees Celsius for 30 minutes. Next, synthesize the first strand of complimentary DNA by setting up a PCR reaction by adding 10.5 microliters of RNA and one microliter of three micrograms per milliliter of random hexamer primers.To denature the secondary structure, first, incubate the tube at 65 degrees Celsius for five minutes, and then on ice for two minutes. Then, add the master mix to the PCR tube and incubate at 25 degrees Celsius for two minutes. Then, add one microliter of 200 units per microliter of superscript two and run the thermocycler program.To synthesize the second strand of complimentary DNA, set up the reaction on ice by adding the reagents, buffer, and enzymes. Then, vortex to mix the reagents gently and incubate at 16 degrees Celsius for two hours. After the DNA has been synthesized, purify the complimentary DNA using a PCR purification kit.Then, defragment the double-stranded DNA, use a water bath sonifier and a 1.5 milliliter tube to share the DNA to a size of 250 to 500 base pairs for three times 10 minutes or four times 10 minutes. Then, prepare the library and perform next generation sequencing. Here, phase contrast images of embryonic stem cells were captured to visualize the stem cell colonies.In this protocol, the cells were cultured in the presence or absence of mitotically inactivated mouse embryonic fibroblasts with LIF and/or GSK3i in the culture media to prevent the differentiation of the cells. Then, to visualize the efficiency of sonication of the cross-linked embryonic stem cell chromatin, sonicated double-stranded DNA was loaded on a 2%agarose gel. The gel image shows sheared chromatin in the range of 200 to 500 base pairs.Next, ChIP-Seq libraries were generated and sequenced using a next generation sequencing platform to evaluate genome-wide occupancy of a histone modifying enzyme and histone modifications in embryonic stem cells. From the custom UCSC browser view, enriched H3K4 trimethylation, H3K4 dimethylation, and KDM5B are visible at the core pluripotency regulator gene, POU5F1 and at the HOXC cluster. CARIP-Seq analysis was also performed to evaluate chromatin-associated RNAs in embryonic stem cells.The custom UCSC browser view shows H4K20 trimethylation associated RNA and H4K20 trimethylation occupancy in embryonic stem cells. From the CARIP-Seq results, it can be interpreted that although the RNA is associated with H4K20 trimethylation, it might not interact with the particular locus. Once mastered, this technique can be done in four days if it is performed properly.After watching this video, you should have a good understanding of how to survey chromatin-associated RNAs using CARIP-Seq and identify protein DNA interactions on a global scale in embryonic stem cells.

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9.7K Views.  Wayne State University School of Medicine. The overall goal of CARIP-Seq is to survey genome-wide chromatin-associated RNAs. The goal of ChIP-Seq is to evaluate protein-DNA interactions on a global scale in embryonic stem cells. These methods can help answer key questions in the epigenetics and stem cell fields, identify the genome location transcription factors or epigenetic regulator binding or histone modification occupancy or chromatin-associated RNAs in embryonic stem cells.The main advantage of these techniques is that th...