Name: Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome (Video) | JoVE Journal
Uploaded: 2023-02-24T12:50:00.000+00:00
Duration: 12 min 25 s
Description: Learn paired epigenomic and transcriptomic analysis of specific ovarian cell populations using optimized TRAP and INTACT methods in mice. This technique enables high-throughput study of cell type-specific changes.

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5 minutes

This work was supported by grants from the National Institutes of Health (NIH) (R01AG070035, R01AG069742, T32AG052363), BrightFocus Foundation (M2020207), and Presbyterian Health Foundation. This work was also supported in part by the MERIT award I01BX003906 and a Shared Equipment Evaluation Program (ShEEP) award ISIBX004797 from the United States (U.S.) Department of Veterans Affairs, Biomedical Laboratory Research and Development Service. The authors would also like to thank the Clinical Genomics Center (OMRF) and Imaging Core Facility (OMRF) for assistance and instrument usage.

All animal procedures were approved by the Institutional Animal Care and Use Committee at the Oklahoma Medical Research Foundation (OMRF). Parent mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and bred and housed under SPF conditions in a HEPA barrier environment on a 14 h/10 h light/dark cycle (lights on at 6:00am) at the OMRF.
NOTE: In this demonstration, we use a Cyp17iCre+/&#8722;(Strain # 028547, The Jackson Laboratory) male paired with a NuTRAP female (Strain # 029899, The Jackson Laboratory). The desired Cyp17-NuTRAP progeny (Cyp17iCre+/&#8722;; NuTRAPflox/WT) will express the NuTRAP allele under the control of the Cyp17a1&#160;promoter in ovarian stromal cells. For this manuscript preparation, we used 4-month-old female Cyp17-NuTRAP mice (n = 4). DNA was extracted from mouse ear punch samples for genotyping to confirm the inheritance of the Cyp17iCre and NuTRAP transgenes and for the PCR detection of 1) generic Cre, 2) Cyp17iCre, and 3) NuTRAP floxed using the primers listed in the Table of Materials, as previously described7,8.
1. Mouse ovarian dissection
<ol>
	<li>Perform mouse euthanasia according to the animal care guidelines and approval of the institution, followed by ovary collection.</li>
	<li>Dissect the ovaries to remove any adipose tissue or parts of the oviducts that might be attached to the ovarian tissue prior to placing the ovaries in tubes with buffer. Immediately proceed to TRAP or INTACT techniques. 
	NOTE: This protocol has not been optimized for use with frozen tissue. Using one ovary from the same mouse for each technique is recommended for future statistical analysis.</li>
</ol>
2. Isolation of nuclei from specific ovarian cell types
<ol>
	<li>Buffer preparation
	<ol>
		<li>Nuclear purification buffer (NPB): To prepare 100 mL of NPB, combine 2 mL of 1 M HEPES (final concentration: 20 mM HEPES), 0.8 mL of 5 M NaCl (40 mM NaCl), 4.5 mL of 2 M KCl (90 mM KCl), 0.4 mL of 0.5 M EDTA (2 mM EDTA), 0.1 mL of 0.5 M EGTA (0.5 mM EGTA), and 92.2 mL of nuclease-free water. Supplement with 1x protease inhibitor on the day of nuclei isolation. 
		NOTE: NPB can be prepared without the protease inhibitor in advance and lasts for up to 3 weeks at 4 &#176;C.</li>
		<li>Nuclei lysis buffer (complete): Supplement nuclei lysis buffer with 1x protease inhibitor on the day of nuclei isolation.</li>
	</ol>
	</li>
	<li>Creation of the nuclei suspension 
	NOTE: The samples should be maintained on ice at all times unless otherwise indicated.
	<ol>
		<li>Collect one ovary from a Cyp17-NuTRAP mouse (or other relevant Cre-NuTRAP) in 500 &#181;L of nuclei lysis buffer (complete). Chop the ovary into eight parts within the buffer using self-opening micro scissors.</li>
		<li>Use wide-bore pipette tips to transfer the minced ovary and 500 &#181;L of lysis buffer into a glass Dounce homogenizer on ice. Homogenize the ovary 10x-20x with the loose pestle A. Add 400 &#181;L of lysis buffer to the Dounce homogenizer, washing pestle A.</li>
		<li>Homogenize the ovary with the tight pestle B 10x-20x. Add 1,000 &#181;L of lysis buffer, washing pestle B. Transfer the homogenate (1.9 mL) to a 2 mL round-bottom tube.</li>
		<li>Centrifuge at 200 x g for 1.5 min at 4 &#176;C to remove undissociated tissue and blood vessels14.The supernatant contains the nuclei; discard the pellet of undissociated tissue.</li>
		<li>After pre-wetting a 30 &#181;m cell strainer with 100 &#181;L of lysis buffer, and filter the supernatant through the cell strainer into a 15 mL conical tube. Then, transfer the nuclei-containing mixture to a 2 mL round-bottom tube.</li>
		<li>Centrifuge the sample at 500 x g for 5 min at 4 &#176;C, and discard the supernatant. The pellet contains nuclei.</li>
		<li>Resuspend the nuclear pellet in 250 &#181;L of ice-cold lysis buffer by vortexing briefly at moderate to high speed. Bring the volume up to 1.75 mL by adding 1.5 mL of lysis buffer. Mix gently, and rest the nuclear suspension on ice for 5 min.</li>
		<li>Pellet the nuclei by centrifugation at 500 x g for 5 min at 4 &#176;C. Carefully discard the supernatant without disturbing the nuclear pellet. Resuspend the pellet in 200 &#181;L of ice-cold storage buffer, and vortex briefly to completely resuspend the nuclear pellet.</li>
		<li>Reserve 10% of the resuspended pellet (20 &#181;L) as an input nuclei sample for downstream analyses.</li>
		<li>Take the resuspended nuclei pellet, and make up the volume to 2.0 mL with ice-cold NPB. Proceed to isolate the labeled nuclei using the INTACT affinity-based purification method.</li>
	</ol>
	</li>
	<li>Isolation of nuclei tagged in specific cell types (INTACT)
	<ol>
		<li>Vortex the streptavidin beads in the vial for 30 s at medium speed to ensure they are fully resuspended. Add 30 &#181;L of resuspended beads for each sample into a 2 mL round-bottom tube (beads for up to 10 samples can be washed in a single tube), add 1 mL of NPB, and resuspend well by pipetting.</li>
		<li>Place the tube on the magnetic rack for 1 min to separate the beads. Discard the supernatant, and remove the tube from the magnet. Resuspend the washed magnetic beads in 1 mL of NPB.</li>
		<li>Repeat the wash step for a total of three washes. Following the final wash, resuspend the beads in the initial volume of NPB (30 &#181;L per sample).</li>
		<li>Add 30 &#181;L of the washed beads to the 2 mL nuclear suspension from step 2.2.10. Resuspend the bead-nuclei mixture well by pipetting and gently inverting the tube.</li>
		<li>Place the tubes on a rotating mixer in a cold room or refrigerator for 30 min at low speed. Incubate the input samples without beads in the same conditions.</li>
		<li>Place the tubes with the nuclear suspension and beads on the magnet, and separate the biotinylated nuclei bound to streptavidin beads (positive fraction) from the negative fraction of nuclei. Allow the beads to separate on the magnet for 3 min.</li>
		<li>Remove the supernatant, pass the negative fraction to a fresh 2.0 mL tube, and reserve on ice. For each positive fraction tube, remove the tube from the magnet, and resuspend the contents in 1 mL of NPB.</li>
		<li>Place the tube on the magnet for 1 min, and discard the supernatant. Remove the tube from the magnet, and resuspend the washed bead-nuclei mixture in 1 mL of NPB.</li>
		<li>Repeat step 2.3.8 for a total of three washes. After the completion of three washes, resuspend the positive fraction bead-nuclei mixture in 30 &#181;L of NPB.</li>
		<li>For each negative fraction tube, centrifuge the nuclear suspension from step 2.3.7 at 500 x g for 7 min at 4 &#176;C. Carefully aspirate and discard the supernatant. Resuspend the negative fraction nuclei pellet in 30 &#181;L of NPB.</li>
		<li>Store the nuclei from the input (step 2.2.9), the negative fraction (step 2.3.10), and the positive fraction (step 2.3.9) in a &#8722;80 &#176;C freezer until ready for nuclear DNA or RNA isolation. 
		NOTE: The nuclei should not be frozen for protocols requiring intact nuclei as input (i.e., assay for transposase-accessible chromatin, chromatin immunoprecipitation).</li>
	</ol>
	</li>
</ol>
3. Isolation of mRNA from specific ovarian cell types
<ol>
	<li>Preparing the buffers
	<ol>
		<li>TRAP buffer base: Prepare 100 mL of TRAP buffer base by combining 78.8 mL of RNase-free water, 5 mL of 1 M Tris-HCl (final concentration: 50 mM Tris [pH 7.5]), 5 mL of 2 M KCl (100 mM KCl), 1.2 mL of 1 M MgCl2 (12 mM MgCl2), and 10 mL of 10% NP-40 (1% NP-40). Store at 4 &#176;C for up to 1 month.</li>
		<li>High salt buffer base: Prepare 100 mL of high salt buffer base by combining 68.8 mL of RNase-free water, 5 mL of 1 M Tris-HCl (final concentration: 50 mM Tris [pH 7.5]), 15 mL of 2 M KCl (300 mM KCl), 1.2 mL of 1M MgCl2 (12 mM MgCl2), and 10 mL of 10% NP-40 (1% NP-40). Store at 4 &#176;C for up to 1 month.</li>
		<li>TRAP homogenization buffer (complete): Prepare 1.5 mL per sample on the day of tissue collection. Prepare 10 mL of TRAP homogenization buffer by combining 10 mL of TRAP buffer base (from step 3.1.1) with 10 &#181;L of 100 mg/mL cycloheximide (final concentration: 100 &#181;g/mL cycloheximide), 10 mg of sodium heparin (1 mg/mL sodium heparin), 20 &#181;L of 0.5 M DTT (1 mM DTT), 50 &#181;L of 40 U/&#181;L RNase inhibitor (200 U/mL RNase inhibitor), 200 &#181;L of 0.1 M spermidine (2 mM spermidine), and one EDTA-free protease-inhibitor tablet (1x).</li>
		<li>Low salt wash buffer (complete): Make 1.5 mL per sample on the day of tissue collection. To make 10 mL of low salt wash buffer, combine 10 mL of TRAP buffer base (from step 3.1.1) with 10 &#181;L of 100 mg/mL cycloheximide (100 &#181;g/mL cycloheximide) and 20 &#181;L of 0.5 M DTT (1 mM DTT).</li>
		<li>High salt wash buffer (complete): Make 1.5 mL per sample on the second day of TRAP isolation. To make 10 mL of high salt wash buffer, combine 10 mL of high salt buffer base (from step 3.1.2) with 10 &#181;L of 100 mg/mL cycloheximide and 20 &#181;L of 0.5 M DTT.</li>
	</ol>
	</li>
	<li>Performing translating ribosome affinity purification (TRAP)
	<ol>
		<li>Collect the other ovary from a Cyp17-NuTRAP mouse (or other relevant Cre-NuTRAP) and place it in a 1.5 mL RNase-free tube containing 100 &#181;L of ice-cold TRAP homogenization buffer (from step 3.1.3). Chop the ovary into eight parts within the buffer using self-opening micro scissors.</li>
		<li>Use wide-bore tips to transfer the ovary and 100 &#181;L of homogenization buffer into a glass Dounce homogenizer. Homogenize 10x-20x with the loose pestle A. Add 400 &#181;L of TRAP homogenization buffer, washing pestle A.</li>
		<li>Transfer the homogenate to a 2 mL round-bottom tube. Wash the Dounce homogenizer with an additional 1 mL of TRAP homogenization buffer, and transfer the washed volume to the 2 mL round-bottom tube.</li>
		<li>Centrifuge the homogenate at 12,000 x g for 10 min at 4 &#176;C. Transfer the cleared supernatant to a fresh 2 mL round-bottom tube. Discard the pellet.</li>
		<li>Transfer 100 &#181;L of the cleared homogenate to a fresh 2 mL round-bottom tube, and reserve as input on ice.</li>
		<li>Incubate the remainder of the cleared homogenate (from step 3.2.4) with an anti-GFP antibody (5 &#181;g/mL) for 1 h at 4 &#176;C while rotating end-over-end. Incubate the input sample in the same conditions.</li>
		<li>In the last 15 min of the incubation, wash the protein G magnetic beads in the low salt wash buffer using the following steps (3.2.8-3.2.11).</li>
		<li>Vortex the vial of protein G magnetic beads for 30 s to completely resuspend. Transfer 50 &#181;L of the protein G magnetic beads for each sample (up to 10 samples can be washed in one tube) into a 2 mL round-bottom tube.</li>
		<li>Add 500 &#181;L of low salt wash buffer to the beads, and pipette gently to mix. Place the tube into a magnetic stand for 1 min to collect the beads against the side of the tube. Remove and discard the supernatant.</li>
		<li>Remove the tube from the magnetic stand, and add 1 mL of low salt wash buffer to the tube. Pipette gently to mix. Place the tube into a magnetic stand for 1 min to collect the beads against the side of the tube. Remove and discard the supernatant.</li>
		<li>Repeat step 3.2.10 for a total of three washes. Following the final wash, remove the tube from the magnetic rack, and resuspend the beads in the initial volume of low salt wash buffer (50 &#181;L per sample).</li>
		<li>Transfer the resuspended washed beads (50 &#181;L) to the antigen sample/antibody mixture, and incubate at 4 &#176;C overnight while rotating in an end-over-end mixer. Incubate the input samples rotating end-over-end at 4 &#176;C overnight to maintain equivalent conditions.</li>
		<li>After the overnight incubation, remove the tubes from the rotator, and separate the magnetic beads with the target ribosomes/RNA (positive fraction) from the supernatant (negative fraction) using the magnetic stand for 2.5-3 min. Aspirate the negative fraction, place it in a fresh 2 mL round-bottom tube, and set it aside on ice while processing the positive fraction.</li>
		<li>Add 500 &#181;L of high salt wash buffer (from step 3.1.5) to the tube with the positive fraction, and gently pipette to mix. Place the tube on a magnetic stand maintained on ice for 1 min to separate the beads. Discard the supernatant, and remove the tube from the magnet.</li>
		<li>Repeat step 3.2.14 for a total of three washes. Following the final wash, resuspend the beads in 350 &#181;L of RNA lysis buffer supplemented with 3.5 &#181;L of 2-mercaptoethanol (BME). Incubate the tubes at room temperature while mixing for 10 min at 900 rpm in a digital shaker.</li>
		<li>Separate the beads from the solution that now contains the target ribosomes/RNA with a magnetic stand for 1 min at room temperature. Collect the eluted positive fraction (supernatant) in a fresh 1.5 mL tube, and add 350 &#181;L of 100% ethanol. Invert to mix. Proceed to the RNA isolation.</li>
		<li>Optional: To isolate RNA from the input sample (step 3.2.5) and negative fraction (step 3.2.13), transfer 100 &#181;L of the input and negative fractions to fresh 1.5 mL tubes. Add 350 &#181;L of RNA lysis buffer supplemented with 3.5 &#181;L of BME to each sample. Invert to mix. Add 250 &#181;L of 100% ethanol to each tube, and invert to mix. Proceed to the RNA isolation.</li>
	</ol>
	</li>
	<li>RNA isolation
	<ol>
		<li>Transfer 700 &#181;L of the positive fraction and, optionally, the input and/or negative fraction to an RNA spin column. Follow the manufacturer&#39;s instructions to isolate RNA from the spin column.</li>
	</ol>
	</li>
</ol>

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<li>Natarajan, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+tagged+reverse+transcription+%28STRT-Seq%29%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Single-cell tagged reverse transcription (STRT-Seq).</a> Methods in Molecular Biology. 1979, 133-153 (2019).</li>
<li>Jaitin, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Massively+parallel+single-cell+RNA-seq+for+marker-free+decomposition+of+tissues+into+cell+types%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Massively parallel single-cell RNA-seq for marker-free decomposition of tissues into cell types.</a> Science. 343 (6172), 776-779 (2014).</li>
<li>Aldridge, S., Teichmann, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+cell+transcriptomics+comes+of+age%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Single cell transcriptomics comes of age.</a> Nature Communications. 11, 4307(2020).</li>
<li>Clark, S. J., Lee, H. J., Smallwood, S. A., Kelsey, G., Reik, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+epigenomics%3A+Powerful+new+methods+for+understanding+gene+regulation+and+cell+identity%5BTitle%5D)+AND+%22Genome+Biology%22%5BJournal%5D)">Single-cell epigenomics: Powerful new methods for understanding gene regulation and cell identity.</a> Genome Biology. 17, 72(2016).</li>
<li>Christensson, E., Lewan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+spermidine+for+the+isolation+of+nuclei+from+mouse+liver.+Studies+of+purity+and+yield+during+different+physiological+conditions%5BTitle%5D)+AND+%22Zeitschrift+fu%CC%88r+Naturforschung.+Section+C%2C+Biosciences%22%5BJournal%5D)">The use of spermidine for the isolation of nuclei from mouse liver. Studies of purity and yield during different physiological conditions.</a> Zeitschrift für Naturforschung. Section C, Biosciences. 29 (5-6), 267-271 (1974).</li>
<li>Levine, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Menopause+accelerates+biological+aging%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Menopause accelerates biological aging.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (33), 9327-9332 (2016).</li>
<li>Ossewaarde, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age+at+menopause%2C+cause-specific+mortality+and+total+life+expectancy%5BTitle%5D)+AND+%22Epidemiology%22%5BJournal%5D)">Age at menopause, cause-specific mortality and total life expectancy.</a> Epidemiology. 16 (4), 556-562 (2005).</li>
<li>Wellons, M., Ouyang, P., Schreiner, P. J., Herrington, D. M., Vaidya, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+menopause+predicts+future+coronary+heart+disease+and+stroke%3A+the+Multi-Ethnic+Study+of+Atherosclerosis%5BTitle%5D)+AND+%22Menopause%22%5BJournal%5D)">Early menopause predicts future coronary heart disease and stroke: the Multi-Ethnic Study of Atherosclerosis.</a> Menopause. 19 (10), 1081-1087 (2012).</li>
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The authors declare no conflicts of interest.

The NuTRAP mouse model6&#160;is a powerful transgenic labeling approach for the paired interrogation of the transcriptome and epigenome from specific cell types that can be adapted to any cell type with an available Cre driver. Here, we demonstrate the specificity of the Cyp17-NuTRAP mouse model in targeting ovarian theca and stromal cells. The Cyp17-NuTRAP model can be used to further elucidate the theca and stromal cell-specific epigenetic mechanisms involved in ovarian aging, cancer, and diseas...

The ovaries are major players in somatic aging1, with distinct contributions from specific cell populations. The cellular heterogeneity of the ovary makes it difficult to interpret molecular results from bulk, whole-ovary assays. Understanding the role of specific cell populations in ovarian aging is key to identifying the molecular drivers responsible for fertility and health decline in aged women. Traditionally, the multi-omics assessment of specific ovarian cell types was achieved by techniques such as laser microdissection2, single-cell approaches3, or cell sorting4. However, microdissection can be expensive and difficult to perform, and cell sorting can alter cellular phenotypic profiles5.
A novel approach to assess ovarian cell-type-specific epigenomic and transcriptomic profiles uses the nuclear tagging and translating ribosome affinity purification (NuTRAP) mouse model. The NuTRAP model allows the isolation of cell-type-specific nucleic acids without the need for cell sorting by using the affinity purification methods: translating ribosome affinity purification (TRAP) and isolation of nuclei tagged in specific cell types (INTACT)6. The expression of the NuTRAP allele is under the control of a floxed STOP cassette and can be targeted to specific ovarian cell types using promoter-specific Cre lines. By crossing the NuTRAP mouse with a cell-type-specific Cre line, the removal of the STOP cassette causes eGFP-tagging of the ribosomal complex and biotin/mCherry-tagging of the nucleus in a Cre-dependent manner6. The TRAP and INTACT techniques can then be used to isolate mRNA and nuclear DNA from the cell type of interest and proceed to transcriptomic and epigenomic analyses.
The NuTRAP model has been used in different tissues, such as adipose tissue6, brain tissue7,8,9, and the retina10, to reveal cell-type-specific epigenomic and transcriptomic changes that may not be detected in whole-tissue homogenate. The benefits of the NuTRAP approach over traditional cell sorting techniques include the following: 1) the prevention of ex vivo activational artifacts8, 2) the minimized need for specialized equipment (i.e., cell sorters), and 3) the increased throughput and decreased cost of cell-type-specific analyses. In addition, the ability to isolate cell-type-specific DNA and RNA from a single mouse allows for paired analyses that increase the statistical power. Since recent studies have implicated ovarian stromal cells in driving premature aging phenotypes11,12,13, we targeted the NuTRAP expression system to stromal and theca cells using a Cyp17a1-Cre driver. Here, we demonstrate that the induction of the NuTRAP construct is specific to ovarian stromal and theca cells, and sufficient DNA and RNA for sequencing studies are obtained from a single ovary. The NuTRAP model and methods presented here can be used to study any ovarian cell type with any available Cre line.
For the generation of a cell-type-specific ovarian NuTRAP mouse line, the nuclear tagging and translating ribosome affinity purification (NuTRAP) allele has a floxed STOP codon that controls the expression of BirA, biotin ligase recognition peptide (BLRP)-tagged mCherry/mRANGAP1, and eGFP/L10a. When crossed with a cell-type-specific Cre line, the expression of the NuTRAP cassette labels the nuclear protein mRANGAP1 with biotin/mCherry and ribosomal protein L10a with eGFP in a Cre-dependent manner. This allows for the isolation of nuclei and mRNA from specific cell types without the need for cell sorting. The NuTRAPflox/flox can be paired with a cell-type-specific Cre relevant to ovarian cell types to assess this.

<table><tbody><tr><td>0.1 M Spermidine</td><td>Sigma-Aldrich</td><td>05292-1ML-F</td><td></td></tr><tr><td>1 M MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Thermo Scientific</td><td>AM9530G</td><td></td></tr><tr><td>10% NP-40</td><td>Thermo Scientific</td><td>85124</td><td></td></tr><tr><td>100 mg/mL Cycloheximide</td><td>Sigma-Aldrich</td><td>C4859-1ML</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td></td></tr><tr><td>30 &amp;micro;m cell strainer&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-098-458</td><td></td></tr><tr><td>All Prep DNA/RNA Mini Kit</td><td>Qiagen</td><td>80204</td><td></td></tr><tr><td>anti-GFP antibody</td><td>Abcam</td><td>Ab290</td><td>For TRAP and IHC (Rabbit polyclonal to GFP)</td></tr><tr><td>Buffer RLT</td><td>Qiagen</td><td>79216</td><td>RNA Lysis Buffer in protocol</td></tr><tr><td>cOmplete, mini, EDTA-free protease inhibitor tablet</td><td>Roche</td><td>11836170001</td><td>For TRAP Homogenization Buffer</td></tr><tr><td>Cyp17iCre mouse model</td><td>The Jackson Laboratory</td><td>28547</td><td>B6;SJL-Tg(Cyp17a1-icre)AJako/J</td></tr><tr><td>DynaMag-2 magnet</td><td>Invitrogen</td><td>12321D</td><td></td></tr><tr><td>Genotyping Primers</td><td>IDT</td><td>Custom</td><td>Generic Cre - Jackson Laboratory protocol 22392, Primers: oIMR1084, oIMR1085, oIMR7338, oIMR7339</td></tr><tr><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>Cyp17iCre - Jackson Laboratory protocol 30847, Primers: 21218, 31704, 31705, 35663</td></tr><tr><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>NuTRAP - Jackson Laboratory protocol 21509, Primers: 21306, 24493, 32625, 32626</td></tr><tr><td>Halt Protease Inhibitor cocktail (100X)</td><td>Thermo Scientific</td><td>1861278</td><td>For NPB Buffer</td></tr><tr><td>M-280 Streptavidin Dynabeads&amp;nbsp;</td><td>Invitrogen</td><td>11205D</td><td>2.8 &amp;micro;m bead diameter</td></tr><tr><td>MixMate</td><td>Eppendorf</td><td>5353000529</td><td></td></tr><tr><td>Nuclei Isolation Kit: Nuclei EZ Prep</td><td>Sigma-Aldrich</td><td>Nuc101</td><td>Contains Nuclei Lysis Buffer and Nuclei Storage Buffer</td></tr><tr><td>1 M HEPES</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>5 M NaCl</td><td>Thermo Scientific</td><td>AM9760G</td><td></td></tr><tr><td>2M KCl</td><td>Thermo Scientific</td><td>AM9640G</td><td></td></tr><tr><td>0.5 M EDTA</td><td>Thermo Scientific</td><td>AM9260G</td><td></td></tr><tr><td>0.5 M EGTA</td><td>Fisher Scientific</td><td>50-255-956</td><td></td></tr><tr><td>NuTRAP mouse model</td><td>The Jackson Laboratory</td><td>29899</td><td>B6;129S6-Gt(ROSA)26Sortm2(CAG-NuTRAP)Evdr/J</td></tr><tr><td>Pierce DTT No-Weigh Format</td><td>Thermo Scientific</td><td>A39255</td><td></td></tr><tr><td>Protein G Dynabeads</td><td>ThermoFisher</td><td>10004D</td><td>For TRAP</td></tr><tr><td>RNaseOUT</td><td>Invitrogen</td><td>10777019</td><td></td></tr><tr><td>Sodium Heparin</td><td>Fisher Scientific</td><td>BP2425</td><td></td></tr><tr><td>Ultrapure 1M Tris-HCl, pH 7.5</td><td>Invitrogen</td><td>15567-027</td><td></td></tr><tr><td>VWR Tube Rotator</td><td>Fisher Scientific</td><td>NC9854190</td><td></td></tr></tbody></table>

cell-specific paired interrogation of the mouse ovarian epigenome and transcriptome

Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method was performed for the subsequent paired interrogation of the cell-specific ovarian transcriptome and epigenome using a novel transgenic NuTRAP mouse model. The expression of the NuTRAP allele is under the control of a floxed STOP cassette and can be targeted to specific ovarian cell types using promoter-specific Cre lines. Since recent studies have implicated ovarian stromal cells in driving premature aging phenotypes, the NuTRAP expression system was targeted to stromal cells using a Cyp17a1-Cre driver. The induction of the NuTRAP construct was specific to ovarian stromal fibroblasts, and sufficient DNA and RNA for sequencing studies were obtained from a single ovary. The NuTRAP model and methods presented here can be used to study any ovarian cell type with an available Cre line.

A schematic of the TRAP and INTACT protocols is shown in Figure 1. Here the specificity of the Cyp17-NuTRAP mouse model to ovarian stromal/theca cells is demonstrated by immunofluorescent imaging and RNA-Seq from TRAP-isolated RNA. First, immunofluorescence imaging of the eGFP signal in the ovary and localization of the eGFP signal to the theca and stromal cells were performed. Briefly, 5 µm sections were deparaffinized with a xylene and ethanol gradient. For better eGFP signaling, the eGFP ...

In this protocol, the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method were optimized for the paired interrogation of the cell-specific ovarian transcriptome and epigenome using the NuTRAP mouse model crossed to a Cyp17a1-Cre mouse line.

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the ...

cell-specific-paired-interrogation-mouse-ovarian-epigenome

Research

JoVE Journal

Biology

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation.  Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome.  Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

Single Oocyte Bisulfite Mutagenesis

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Here, we present an optimized protocol for imaging entire ovaries for quantitative and qualitative analyses using whole-mount immunostaining, multiphoton microscopy, and 3D visualization and analysis. This protocol accommodates high-throughput, reliable, and repeatable processing that is applicable for toxicology, clinical diagnostics, and genomic assays of ovarian function.

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

A method for microdissection of the mouse oviduct that allows collection of the individual segments while maintaining RNA integrity is presented. In addition,  non-enzymatic oviductal cell dissociation procedure is described. The methods are appropriate for subsequent gene and protein analysis of the functionally different oviductal segments and dissociated oviductal cells.

Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation

This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

Figure 1: End-to-end framework. The figure illustrates end-to-end framework for multimodal knowledge Graph VISHAM-KG. Please click here to view a larger version of this figure.

Preparation of Meiotic Chromosome Spreads from Mouse Oocytes for Assessment of Synapsis and Recombination

Learn paired epigenomic and transcriptomic analysis of specific ovarian cell populations using optimized TRAP and INTACT methods in mice. This technique enables high-throughput study of cell type-specific changes.

Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome

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