an enhanced crosslinking immunoprecipitation method for efficient identification of protein-bound rna in mouse testes

An Enhanced Crosslinking Immunoprecipitation Method for Efficient Identification of Protein-bound RNA in Mouse Testes

To begin this procedure, harvest about 100 milligrams of testes from mice of appropriate age for each immunoprecipitation experiment. Place the tissues in ice-cold PBS. Using a pair of fine-tip tip tweezers, gently remove the tunica albuginea. Add 3 milliliters of ice-cold PBS to a tissue grinder, and use a loose glass pestle to triturate the tissue by mild mechanical force. Next, transfer the tissue suspension to a cell culture dish, and add ice-cold PBS up to 6 milliliters. Shake the plate quickly so that liquid covers the bottom of the dish evenly.
Cross-link the suspension on ice three times with 400 millijoules per centimeter squared at 254 nanometers. Make sure to mix the suspension between each irradiation, then, collect the suspension in a 15-milliliter conical tube, and centrifuge at 1,200 times g and at 4 degrees for 5 minutes. Remove the supernatant and resuspend the pellet in 1 milliliter of PBS. After this, transfer the suspension to a 1.5-milliliter centrifuge tube. Centrifuge at 1,000 times g and at 4 degrees Celsius for two minutes and discard the supernatant.
First, add 125 microliters of protein A magnetic beads per sample to a fresh centrifuge tube. Place the tube on the magnet to separate the beads from the solution. After 10 seconds, remove the supernatant, and wash the beads twice with 1 milliliter of ice-cold lysis buffer. Resuspend the beads in 100 microliters of cold lysis buffer with 10 micrograms of eCLIP antibody. Rotate the tubes at room temperature for 45 minutes, then, wash the beads twice with 1 milliliter of ice-cold lysis buffer.
To begin, resuspend the tissue pellets in 1 milliliter of cold lysis buffer containing 22 microliters of 50X EDTA-free protein inhibitor cocktail and 11 microliters of RNAse inhibitor. Keep lysing the samples on ice for 15 minutes. Sonicate each sample is outlined in the text protocol. Next, add 4 microliters of DNAse to each tube and mix well. Incubate at 37 degrees Celsius for 10 minutes while shaking at 1,200 RPM. After this, add 10 microliters of diluted RNAse and mix well. Incubate at 37 degrees Celsius for 5 minutes while shaking at 1,200 RPM.
Then, centrifuge at 15,000 times g and at 4 degrees Celsius for 20 minutes to clear the lysate. Carefully collect the supernatant. First, add 1 milliliter of the lysate to the prepared beads and rotate the samples at 4 degrees Celsius for either two hours or overnight. After this, collect the beads with the magnetic stand and discard the supernatant. Wash the beads twice with 900 microliters of high-salt buffer, and then wash the beads twice with 900 microliters of wash buffer.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Tissue Harvesting and UV Crosslinking
<ol>
	<li>Euthanize 2 adult mice using carbon dioxide (CO2) for 1-2 min or until breathing stops. Next, perform cervical dislocation on each mouse.</li>
	<li>Harvest about 100 mg of testes from mice of appropriate age (one adult testis in this study) for each immunoprecipitation experiment, and place the tissues in ice-cold phosphate-buffered saline (PBS).</li>
	<li>Remove the tunica albuginea gently with one pair of fine-tipped tweezers.</li>
	<li>Add 3 mL of ice-cold PBS in a tissue grinder and triturate the tissue by mild mechanical force using a loose glass pestle (type A glass pestle). 
	NOTE: The purpose of this step is not to lyse cells, but to pull apart the tissue. Preservation of cell viability and integrity is important.</li>
	<li>Transfer the tissue suspension to a cell culture dish (10 cm in diameter) and add ice-cold PBS up to 6 mL.</li>
	<li>Shake the plate quickly so that the liquid covers the bottom of the dish evenly. If the tissue is ground properly, evenly distributed seminiferous tubules will be visible, whereas the presence of tissue clumps indicates that tissue dispersion is suboptimal.</li>
	<li>Crosslink the suspension three times with 400 mJ/cm2 at 254 nm on ice. Mix suspension between each irradiation. 
	NOTE: For each new experiment, crosslinking should be optimized.</li>
	<li>Collect the suspension in a 15 mL conical tube and pellet at 1,200 x g for 5 min at 4 &#176;C. Remove the supernatant, resuspend the pellet in 1 mL of PBS, and then transfer the suspension to a 1.5 mL centrifuge tube. Spin at 4 &#176;C and 1,000 x g for 2 min, and remove supernatant.</li>
	<li>At this point, immediately proceed with the rest of the protocol, or snap-freeze the pellets in liquid nitrogen and store them at -80 &#176;C until use.</li>
</ol>
2. Beads Preparation
<ol>
	<li>Add 125 &#181;L of protein A magnetic beads per sample (pellet) to a fresh centrifuge tube. 
	NOTE: Use protein G magnetic beads for mouse antibodies.</li>
	<li>Place the tube on the magnet to separate the beads from the solution. After 10 s, remove the supernatant. Wash beads twice with 1 mL of ice-cold lysis buffer. 
	NOTE: Subsequent separation of protein A magnetic beads follows this step. Lysis buffer composition is 50 mM Tris-HCl, pH 7.5; 100 mM NaCl; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate; 1/50 ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (add fresh).</li>
	<li>Resuspend beads in 100 &#181;L of cold lysis buffer with 10 &#181;g eCLIP antibody. Rotate tubes at room temperature for 45 min. 
	NOTE: The final antibody concentration for immunoprecipitation is 10 &#181;g/mL. If the antibody concentration is unknown, the amount of antibody should be optimized.</li>
	<li>Wash beads twice with 1 mL of ice-cold lysis buffer.</li>
</ol>
3. Tissue Lysis and Partial RNA Digestion
<ol>
	<li>Resuspend tissue pellets in 1 mL of cold lysis buffer (add 22 &#181;L of 50x (EDTA)-free protein inhibitor cocktail and 11 &#181;L of RNase inhibitor to 1 mL of lysis buffer).
	<ol>
		<li>Resuspend two UV-crosslinked pellets and two non-crosslinked pellets per group of experiments: UV-crosslinked-1 pellets for eCLIP library (UV-1); UV-crosslinked-2 pellets for eCLIP library (UV-2); non-crosslinked-1 pellets for eCLIP library as a control (non-UV); non-crosslinked-2 pellets for IgG IP to demonstrate the specificity of antibodies. 
		NOTE: The ideal control for the eCLIP library of the UV-crosslinked wide-type testes is that of the UV-crosslinked knockout testes from mice of the same litter.</li>
	</ol>
	</li>
	<li>Keep lysing the samples on ice for 15 min (to prevent degradation of protein and RNA).</li>
	<li>Sonicate in a digital sonicator at 10% amplitude for 5 min, at 30 s on/30 s off. Always place the sample on ice and clean the probe with nuclease-free water between each sample.</li>
	<li>Add 4 &#181;L of DNase to each tube, and mix well. Incubate for 10 min at 37 &#176;C, shaking at 1,200 rpm.</li>
	<li>Add 10 &#181;L of diluted RNase I (4 U/&#181;L RNase I in PBS), and mix well. Incubate for 5 min at 37 &#176;C, shaking at 1,200 rpm.</li>
	<li>Clear the lysate by centrifugation at 15,000 x g for 20 min (at 4 &#176;C).</li>
	<li>Carefully collect the supernatant. Leave 50 &#181;L of lysate and discard the pellet with it.</li>
	<li>Save input samples as RWB (run for western blot) and RRI (run for RNA isolation). Save 20 &#181;L (2%) of UV-1, UV-2, non-UV, and IgG samples as inputs for RWB gel loading. Save 20 &#181;L (2%) of UV-1 or UV-2 samples as inputs for RRI gel loading.</li>
</ol>
4. Immunoprecipitation
<ol>
	<li>Add 1 mL of the lysate (from step 3.7) to the beads (prepared in section 2) and rotate the samples at 4 &#176;C for 2 h or overnight.</li>
	<li>Collect the beads with a magnetic stand and discard the supernatant. Wash the beads twice with 900 &#181;L of high salt buffer (50 mM Tris-HCl, pH 7.5; 1 M NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate), and then wash beads twice with 900 &#181;L of wash buffer (20 mM Tris-HCl, pH 7.5; 10 mM MgCl2; 0.2% Tween-20). 
	NOTE: For the IgG sample, pause the procedure here and store it on ice in a wash buffer.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse MOV10 antibody</td>
			<td>Proteintech, China</td>
			<td>10370-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse MOV10L1 antibody</td>
			<td>Zheng et al.20109</td>
			<td>polyclonal antisera UP2175</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP Goat Anti-Rabbit IgG</td>
			<td>ABclonal</td>
			<td>AS014</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit IgG</td>
			<td>Beyotime, China</td>
			<td>A7016</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>5242R</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital sonifier</td>
			<td>BRANSON,USA</td>
			<td>BBV12081048A</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-2 Magnet</td>
			<td>Invitrogen,USA</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-light cross-linker</td>
			<td>UVP, USA</td>
			<td>CL-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tubes</td>
			<td>Corning, USA</td>
			<td>430791</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtubes tubes</td>
			<td>AXYGEN , USA</td>
			<td>MCT-150-C</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acid phenol/chloroform/isoamyl alcohol</td>
			<td>Solarbio, China</td>
			<td>P1011</td>
			<td>25:24:01</td>
		</tr>
		<tr>
			<td>AffinityScript Enzyme</td>
			<td>Agilent, USA</td>
			<td>600107</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase I</td>
			<td>Invitrogen, USA</td>
			<td>AM2295</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Inhibitor</td>
			<td>Promega, USA</td>
			<td>N251B</td>
			<td></td>
		</tr>
		<tr>
			<td>RQ1 DNase</td>
			<td>Promega,USA</td>
			<td>M610A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample Reducing Agent</td>
			<td>Invitrogen,USA</td>
			<td>NP0009</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS Solution</td>
			<td>Invitrogen, USA</td>
			<td>15553027</td>
			<td>10%</td>
		</tr>
	</tbody>
</table>

Source: Xu, Q., et al. <a href="https://app.jove.com/v/59681">Enhanced Crosslinking Immunoprecipitation (eCLIP) Method for Efficient Identification of Protein-bound RNA in Mouse Testis.</a> J. Vis. Exp. (2019)
This video demonstrates the enhanced crosslinking immunoprecipitation (eCLIP) method for identifying RNA-binding proteins in mouse testis. eCLIP enables the precise identification of binding sites where RNA-binding proteins (RBPs) interact with their target RNAs.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Tissue ...

Take a freshly harvested mouse testis in a chilled buffer. Remove the tunica albuginea, the fibrous tissue layer.
Triturate the testis to release seminiferous tubules containing germ and Sertoli cells.
Add buffer and subject the tubules to UV&#160; radiation for irreversible crosslinking of RNA with associated proteins, preserving interactions.
Centrifuge and discard the supernatant.
Resuspend the tubules in a lysis buffer containing RNase and protease inhibitors.
Sonicate the tissue, releasing protein-RNA complexes and other intracellular components. The RNase and protease inhibitors inactivate endogenous RNases and proteases.
Add DNase to degrade DNA. Introduce diluted RNase for partial RNA digestion.
Centrifuge and transfer the supernatant containing RNA-protein complexes to an antibody-coated magnetic bead solution with high specificity to the target RNA-binding protein.
Apply a magnetic field to separate the bead-bound protein-RNA complexes. Discard the supernatant.
Wash with buffer. Magnetically separate the protein-RNA complexes and use them for further RNA-protein interaction analysis.

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Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

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Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

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Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

RNA足迹的识别:通过RNA免疫沉淀在串联中的蛋白质复合物,然后测序(RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

An Enhanced Crosslinking Immunoprecipitation Method for Efficient Identification of Protein-bound RNA in Mouse Testes

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An Enhanced Crosslinking Immunoprecipitation Method for Efficient Identification of Protein-bound RNA in Mouse Testes