Name: CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells (Video) | JoVE Journal
Uploaded: 2018-05-25T14:00:00.000+00:00
Duration: 11 min 13 s
Description: Learn CARIP-Seq and ChIP-Seq methods to identify chromatin-associated RNAs and protein-DNA interactions in embryonic stem cells. These techniques enable global epigenomic mapping for stem cell and cancer research.

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5 minutes

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.

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</ol>

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>10&lt;sup&gt;6 &lt;/sup&gt;or 10&lt;sup&gt;7 &lt;/sup&gt;units</td></tr><tr><td>GSK3&amp;beta; 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&#039;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&#039;&amp;rarr;5&#039; 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

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...

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, ...

carip-seq-chip-seq-methods-to-identify-chromatin-associated-rnas

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

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