This work was supported by NIGMS (R01GM11755) to P.W.J and by a training grant fellowship from the National Cancer Institute (NIH) (CA009110) to G.H. and J.H.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Johns Hopkins University. Experiments were performed on wild-type C57BL/6J mice.
1. Harvesting Fetal or Neonatal Ovaries and Preparation of Prophase Chromatin Spreads
<ol>
	<li>To extract embryos at 14.5&#8211;19.5 days post-coitum (dpc) sacrifice the pregnant females via cervical dislocation or CO2 asphyxiation according to IACUC guidelines. 
	NOTE: For postnatal day 1&#8211;5 ovaries skip to step 1.3. Figure 1 summarizes the meiotic prophase stages enriched at different embryonic and post-natal ages.</li>
	<li>Open the abdominopelvic cavity using sterile scissors, making a V-shaped opening. Dissect out the maternal uterine horn, separate the embryos from the placenta, and transfer embryos into 35 mm petri dishes containing 3 mL of pre-warmed 1x phosphate buffer saline (PBS) at 37 &#176;C. 
	NOTE: A fliptop incubator set at 37 &#176;C can be used to maintain temperature. In addition, a temperature regulated glass stage can also be used to maintain temperature during ovary manipulation.</li>
	<li>With 3.5 inch surgical scissors, sacrifice embryos or pups via decapitation according to IACUC guidelines. Place decapitated embryos or pups in pre-warmed PBS prior to further dissection.</li>
	<li>Dissect one embryo or pup at a time in a separate 35 mm petri dish containing 3 mL of pre-warmed PBS at 37 &#176;C. Proceed by cutting along the ventral midline of the posterior half of the embryo, along the anterior half below the forelimbs and directly above the hind-limbs and tail as outlined in Figure 2A&#8211;B.</li>
	<li>Open the abdomen using 3.5 inch surgical scissors. Using fine-tipped forceps displace or remove the liver and loops of bowel, exposing the ovaries in a new 35 mm petri dish containing 3 mL of pre-warmed PBS at 37 &#176;C (see guide in Figure 2B). The ovaries are located immediately below and behind the kidney towards the posterior wall of the peritoneal cavity (Figure 2B&#8211;C). A guide for differentiating between male and female gonads is provided in Figure 2D. 
	NOTE: For optimal conditions, rapidly dissect out the ovaries after pregnant female is sacrificed.</li>
	<li>Remove both ovaries from each female fetus using a pair of fine-tipped forceps under a dissection scope and place in a watch glasses or separate wells of a multi-well plate containing 0.5 to 1.0 mL of pre-warmed PBS and maintain at 37 &#176;C.</li>
	<li>Place each pair of ovaries in 0.5 mL hypo-extraction buffer (17 mM trisodium citrate dihydrate, 50 mM sucrose, 5 mM EDTA, 0.5 mM DTT, 30 mM Tris-HCl, protease inhibitor, pH 8.2) in a clean watch glass or a small well of a 9-well plate, making sure to immerse the ovaries completely. Incubate for at least 15 min, but no more than 30 min. 
	NOTE: Make hypo-extraction buffer fresh and use within 2 h of DTT addition.</li>
	<li>During incubation, using a hydrophobic barrier PAP pen, draw two 22 x 22 mm2 squares on a clean glass 25 x 75 mm2, 1 mm thick microscope slide as shown in Figure 3A. 
	NOTE: Slides can be prepared ahead of time.</li>
	<li>Pipette 45&#8211;50 &#181;L of 100 mM sucrose onto a clean slide and transfer one pair of ovaries to the drop.</li>
	<li>Using the sharp ends of two 27 G needles, tease the ovaries apart to release cells into the sucrose solution. With forceps, remove large pieces of ovary and carefully pipette sucrose solution to disperse cells.</li>
	<li>Place 40 &#181;L of fixative solution (1% paraformaldehyde (PFA), 0.2% detergent (see Table of Materials), pH 9.2) into each square of the prepared slide. With a 200 &#181;l pipette tip, spread the fixative solution evenly over the slide surface.</li>
	<li>Pipette 20 &#181;L of the sucrose mix onto the each square of the slide containing the fixative. 
	NOTE: This equates to using one pair of ovaries per slide.</li>
	<li>Incubate the slides in a closed humid chamber overnight at room temperature. 
	NOTE: The overnight incubation can range around (12-15 hrs) at room temperature.</li>
	<li>Open the chamber lid and allow the slides to air-dry completely.</li>
	<li>Wash slides in a Coplin jar containing 50 mL of 0.4% wetting agent-PBS solution (see Table of Materials) for 2 min, air dry, and proceed to immunolabelling protocol (step 6). 
	NOTE: It may be possible to store the slides at -80 &#176;C for later use, but this varies depending on the protein of interest. For optimal results proceed immediately to the immunolabelling protocol (step 6).</li>
</ol>
2. Metaphase I Oocyte Collection
<ol>
	<li>To maximize the number of antral follicles isolated from each mouse, intraperitoneally inject sexually mature virgin female mice with 5 IU of pregnant mare&#39;s serum gonadotropin (PMSG, also known as equine chorionic gonadotropin (eCG)). 
	NOTE: For optimal oocyte yield, mice should be 1 to 3 months of age, as the number of oocytes harvested will decrease with age. To prepare PMSG, dissolve bottle of 5,000 IU in 100 mL of sterile PBS (5 U/0.1 mL) Store in single-use aliquots of 600 &#181;L at -20 &#176;C.</li>
	<li>After 44&#8211;48 h, prepare collection medium, minimum essential medium alpha (MEM&#945;) medium supplemented with 5% fetal bovine serum (FBS) and 3 mg/mL bovine serum albumin (BSA; MEM&#945;/BSA/FBS). Sterilize media through a 0.2 &#181;m pore filter. Decant 2.5 mL of MEM&#945;/BSA/FBS medium into one glass dish per mouse and warm to 37 &#176;C in a 5% CO2 incubator. 
	NOTE: Other collection and culture media that are commercially available (M2 and M16) are also commonly used. 
	Caution: We recommend removing culture dishes from the 5% CO2 incubator one at a time for limited duration to minimize ambient air exposure during manipulation steps.</li>
	<li>Sacrifice the mice via cervical dislocation or CO2 asphyxiation according to IACUC guidelines.</li>
	<li>Open the abdominopelvic cavity using sterile scissors, making a large V-shaped opening. Using forceps, displace intestines towards the head of the mouse. Locate each uterine horn; the ovaries are present proximal to the rib cage. Hold the oviduct with fine forceps and cut the fat superior to the ovary using dissection scissors (Figure 4). Continue to hold the oviduct, and with another set of fine forceps release the ovary from the bursa and place into collection dish containing MEM&#945;/BSA/FBS medium. 
	NOTE: As in step 1, it is imperative to keep the ovaries at 37 &#176;C and maintain at 5% CO2. A fliptop incubator hooked up to 5% CO2 mix can be used to allow for optimal maintenance of temperature and CO2 levels. In addition, a temperature regulated glass stage should also be used to maintain temperature during oocyte manipulation.</li>
	<li>Using a 1 mL syringe with a 27-gauge needle (or similar size), release cumulus oocyte complexes by manually puncturing the large antral follicles.</li>
	<li>While observing through a dissection microscope, collect GV-staged oocytes using a mouth-operated glass pipette or capillary, or a hand-operated micrometer-syringe (Figure 5). Only collect oocytes that are released from antral follicles. GV oocytes have a diameter of approximately 90 &#181;m. GV oocytes may be surrounded by 2-3 layers of granulosa cells, and have a total diameter of around 200 &#181;m.</li>
	<li>Culture oocytes in a clean watch glass or a 9-well plate containing MEM&#945;/BSA/FBS medium for 6 h to reach metaphase I at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Proceed to step 4.</li>
</ol>
3. Metaphase II (MII) Oocyte Collection
<ol>
	<li>To maximize oocytes isolated from each mouse, intraperitoneally inject sexually mature virgin female mice with 5 IU of PMSG. After 44&#8211;48 h, intraperitoneally inject with 5 IU of human chorionic gonadotropin (hCG). 
	NOTE: For optimal oocyte yield, mice should be 1 to 3 months of age, as the number of oocytes harvested will decrease with age. To prepare hCG, dissolve a bottle of 10,000 U in 200 mL PBS (5 U/0.1 mL). Store in single-use aliquots of 600 &#181;L at -20 &#176;C.</li>
	<li>After 12-14 hrs, prepare MEM&#945;/BSA/FBS collection medium, as described in step 2.2, above.</li>
	<li>Sacrifice the mice via cervical dislocation or CO2 asphyxiation according to IACUC guidelines.</li>
	<li>Open the abdominopelvic cavity using sterile scissors, making a large V-shaped opening. Using forceps, displace intestines towards the head of the mouse. Locate each uterine horn, the ovaries are present proximal to the rib cage. Hold the oviduct with fine forceps and cut the fat superior to the ovary using dissection scissors (Figure 4). Then remove the ovary and oviduct and place into collection dish containing MEM&#945;/BSA/FBS medium. 
	NOTE: As in steps 1 and 2, it is imperative to keep the ovaries at 37 &#176;C and maintain at 5% CO2. A fliptop incubator hooked up to 5% CO2 mix can be used to allow for optimal maintenance of temperature and CO2 levels. In addition, a temperature regulated glass stage should also be used to maintain temperature during oocyte manipulation.</li>
	<li>Using a 1 mL syringe with a 27 G (or similar size) or sharp forceps, tear a hole in the ampulla of the oviduct to release the MII oocytes. 
	NOTE: Be careful not to damage the oocytes in the ampulla. The ampulla will appear swollen and translucent such that the oocytes are visible (Figure 4).</li>
	<li>Harvest MII oocytes from the ampulla of the oviduct into a new dish with collection medium using a mouth-operated glass pipette or capillary, or a hand-operated micrometer-syringe (Figure 5).</li>
	<li>Proceed to step 4.</li>
</ol>
4. Oocyte Denuding and Zona Pellucida Removal
<ol>
	<li>Prepare 300 IU/mL of hyaluronidase in MEM&#945; medium supplemented with 3 mg/mL BSA to denude oocytes of surrounding cumulus cells (2.5 mL/mouse) in a watch glass, and keep at 37 &#176;C, 5% CO2. 
	NOTE: Hyaluronidase efficacy sharply decreases after 1 h of preparation. For MII oocytes, hyaluronidase treatment is not required.</li>
	<li>Expose oocyte-cumulus cell complexes to 300 IU/mL of hyaluronidase in MEM&#945;/BSA for 3 min to denude oocytes of surrounding cumulus cells. 
	Caution: Do not exceed 3 min exposure to hyaluronidase, as it will damage the oocytes.</li>
	<li>To wash the oocytes, transfer oocytes to a fresh warmed MEM&#945;/BSA medium dish. Using a small mouth-operated glass pipette or capillary (slightly larger than the diameter of the oocyte, which is approximately 90 &#181;m), pipette the complexes up and down to completely detach the cumulus cells. Allow the oocytes to recover in the incubator while preparing for the next step.</li>
	<li>Warm acidic Tyrode&#39;s solution, MEM&#945;/BSA, and Waymouth&#39;s media to 37 &#176;C (500 &#181;L/mouse). Place 300 &#181;L in each well of small 9-well glass plates.</li>
	<li>To remove the zona pellucida (ZP) transfer 5&#8211;10 oocytes into Tyrode&#39;s solution. Expose the oocytes for only 30-45 s to the solution. 
	NOTE: Too little exposure to the solution will not remove the ZP completely, which will prevent the oocytes from bursting and chromosomes from spreading. Too much exposure will damage and kill the oocyte. Watching the ZP dissolve under the dissection microscope can aid in optimizing exposure time. 
	Caution: Using fresh Tyrode&#39;s solution is critical, as its function declines with age. Use a fresh well of Tyrode&#39;s solution for each group of oocytes treated to ensure uniform digestion of the ZP.</li>
	<li>Immediately after ZP removal, wash the oocytes by transferring into warmed MEM&#945;/BSA/FBS medium. Repeat this wash step. 
	NOTE: Be careful when transferring, as oocytes will easily stick together and to the glass pipette or capillary following removal of the ZP. Pre-wetting the glass pipette or capillary in Waymouth&#39;s medium will minimize oocytes sticking together.</li>
	<li>Transfer and allow the oocytes to recover for 30 minutes in Waymouth&#39;s medium at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Proceed to step 5.</li>
</ol>
5. MI and MII Oocyte Chromatin Spreads
<ol>
	<li>Using a PAP pen, draw a 11 x 44 mm2 rectangle on the glass slide as shown in Figure 6A.</li>
	<li>Coat the slide with a thin layer of fixative (1% paraformaldehyde, 0.2% Triton-X 100 in H2O, pH 9.2) by pipetting 100 &#181;L of fixative onto the slide and rocking the slide back and forth. Tap the slide to get rid of excess fixative.</li>
	<li>Pick up between 5-10 oocytes with a small mouth pipette needle with as little media as possible and drag the needle in a line across the fixative-coated slide while depositing the oocytes from 1 cm above the slide into the fixative solution. Perform this step using a dissection microscope to ensure that the oocytes have been deposited and that they have burst. 
	NOTE: The oocytes should burst immediately on the slide. The height at which the oocytes are deposited impact the degree of chromatin spreading. See representative results in Figure 6 for further details.</li>
	<li>Incubate slides at room temperature in a closed humidified chamber overnight to allow the chromatin to adhere.</li>
	<li>Allow the slides to air-dry completely and rinse slides in a Coplin jar containing 50 mL of 0.4% wetting agent-H2O, pH 8.0.</li>
	<li>Proceed to step 6.</li>
</ol>
6. Immunolabelling
<ol>
	<li>Prepare the antibody dilution buffer (ADB) described in Table 1.</li>
	<li>Prepare two Coplin jars containing 50 mL of wash buffer (WB) solution (10% ADB diluted in PBS), and one Coplin jar containing 50 mL WB and 0.05% detergent solution (e.g., add 250 &#181;L of 10% detergent to 50 mL WB).</li>
	<li>Wash the slides that were prepared in section 1 and 5 of this protocol for 10 min in one Coplin jar containing 50 mL of WB. 
	Caution: Do not let the slides dry at any point during immunolabelling.</li>
	<li>Wash the slides for 10 min in the Coplin jar containing 50 mL WB and 0.05% detergent solution. Then, wash the slides in the remaining Coplin jar containing 50 mL of WB solution for 10 min.</li>
	<li>Tap off excess liquid and cover slide with 100 &#181;L of selected primary antibodies diluted in ADB. Incubate at 4 &#176;C overnight in a closed humid chamber. 
	NOTE: Incubation can be shortened to 2 to 3 hrs at 37 &#176;C. Use smaller volumes of antibody (e.g., 50 &#181;L) by covering the slide with a coverslip or parafilm.</li>
	<li>Rinse slides in a Coplin jar containing 50 mL of 0.2% wetting agent-PBS solution, pH 8.0.</li>
	<li>Repeat steps 6.2 to 6.5.</li>
	<li>Tap off excess liquid and cover slide with 100 &#181;L of selected secondary antibodies diluted in ADB. Incubate slides 1 to 2 hrs at 37 &#176;C in a closed humid chamber.</li>
	<li>Wash slides 2 times for 10 min in Coplin jars containing 50 mL of 0.2% wetting agent-PBS solution, pH 8.0.</li>
	<li>Wash slides 2 times for 5 min in Coplin jars containing 50 mL of 0.2% wetting agent-H2O solution, pH 8.0.</li>
</ol>
7. Mounting slides
<ol>
	<li>For prophase chromatin spreads prepared in step 2, add 100 &#181;L of mounting medium containing 4&#39;, 6-diamidino-2-phenylindole (DAPI, 1.5&#181;g/mL). For MI and MII chromatin spreads prepared in step 5, add 50 &#181;L of mounting medium containing DAPI (1.5&#181;g/mL). Gently blot away excess liquid.</li>
	<li>Place a 22 mm x 60 mm coverslip on top and seal with clear nail polish. Store in a slide box at 4 &#176;C or -20 &#176;C until assessment via fluorescence microscopy. 
	NOTE: Tap lightly on cover slip to rid of excess mounting medium before sealing with nail polish.</li>
</ol>

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The authors have nothing to disclose.

Chromatin spread preparations allow researchers to chronologically study female mammalian meiosis and the dynamic localization of proteins involved. The embryonic and neonatal chromatin spreads allow for close analysis of events throughout meiotic prophase. Metaphase I and metaphase II chromatin spreads can be used to distinguish single sister chromatids from paired sisters and paired homologous chromosomes, as well as assess ploidy. By comparison, the protocol described here can be advantageous compared to whole oocyte ...

During spermatogenesis, large semi-synchronous waves of meiotic germ cells are rapidly and continuously replenished in the testis at the onset of puberty and throughout adulthood1. In contrast to males, meiosis in females is initiated solely during fetal development. Following birth, oocytes remain arrested in a prolonged dictyate stage of prophase I with an intact germinal vesicle (GV; nuclear envelope) until puberty. At the onset of puberty, a subset of oocytes are cyclically selected to undergo growth and maturation, marking the initiation of meiotic resumption. Meiotic resumption in fully-grown oocytes is manifested by the disappearance of the GV in a process known as germinal vesicle breakdown (GVBD). The oocyte then undergoes chromosome condensation and segregation, followed by polar body extrusion. Oocytes become arrested upon progression to MII and are stimulated to complete the second and final meiotic division only after fertilization.
Female fertility is highly dependent on the success of meiotic prophase I progression. Key to this is formation of a physical linkage between homologous chromosomes known as the chiasmata, which is mediated by repair of induced DNA double strand breaks (DSBs) via crossover recombination2. This process occurs within the context of a dynamic protein-rich scaffold known as the synaptonemal complex (SC) that forms between homologous chromosomes to facilitate their synapsis3. The SC is a zipper-like tripartite structure that consists of two parallel lateral elements connected by central region proteins that holds homologs together throughout ongoing DNA repair. Prior to synapsis, precursors of the lateral elements, called axial elements, form between sister chromatids. Synaptonemal complex proteins such as SYCP2 and SYCP3 form axial elements that colocalize to the sister-chromatid cohesion axes during early prophase. These later serve as binding sites for the transverse filament protein, SYCP1, which facilitates central element assembly and synapsis between aligned homologs4. In mouse oocytes, complete synapsis is indicated by the presence of 20 completely overlapping SYCP3 and SYCP1 stretches, which can be visualized by using chromatin spread preparations. Synapsis is completed upon entry into the pachytene substage, whereby mature crossovers that are destined to form chiasmata between homologs are decorated with mutL homolog (MLH1/3) dimers to promote their accurate processing5,6,7. The structural maintenance of chromosomes (SMC) complexes, including cohesin, condensin, and the SMC5/6 complex, are important for the regulation of chromosome dynamics and structure throughout meiosis8,9,10,11,12. Collectively, these events ensure proper bi-orientation of homologous chromosomes to opposing spindle poles following disassembly of the SC.
The meiotic cell cycle is a powerful model to examine the roles of various proteins in genome maintenance due to the programmed induction and subsequent repair of DNA DSBs. Furthermore, mammalian meiosis is also a relevant model for the study of epigenetic modifications and imprinting13. However, it is technically difficult to assess these events during female meiosis, which takes place in fetal and neonatal ovaries in mammals (Figure 1). Prophase I can be divided into 5 substages: leptonema, zygonema, pachynema, diplonema and dictyate. Herein, we describe how to isolate and distinguish between fetal and neonatal ovaries and testes (Figure 2). Adapted from previously described methods, this manuscript also outlines a protocol (step 1) with video demonstration for preparation of female meiotic prophase I chromatin spreads14,15,16,17. When coupled with immunolabelling, as described in steps 6-7, this protocol enables detailed microscopic analysis of prophase I events in oocytes.
Oogenesis is error-prone, and chromosome missegregation events during the first meiotic division represent the most common source of genetic disease in progeny18,19. In this manuscript (protocol step 2), we describe a protocol in which mature GV-staged oocytes are extracted from primed ovaries of adult female mice. Under supportive conditions, fully-grown oocytes undergo luteinizing hormone-independent resumption of meiosis following isolation and culture20. Following meiotic resumption, oocytes progress through meiosis I, then arrest at metaphase II. Oocytes remain arrested at metaphase II, unless fertilized. In steps 2&#8211;5, we adapt previously reported protocols with video demonstration to describe how to collect, culture and prepare MI and MII oocytes for chromatin spread preparations21. This chromatin spreading technique allows for clear immunolabelling of proteins associated with chromosomes. Furthermore, this protocol can also be used to distinguish between bivalent and univalent chromosomes, and can further resolve single sister chromatids to facilitate assessment of oocyte ploidy. Therefore, in addition to revealing localization patterns of meiotic proteins, this protocol can also serve as an invaluable tool for elucidating potential causes of chromosome missegregation during MI and MII.

<table>
	<tbody>
		<tr>
			<td>10X Phosphate buffered saline (PBS) pH 7.4</td>
			<td>Quality Biological</td>
			<td>119-069-161</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm x 15 mm petri dishes</td>
			<td>Denville</td>
			<td>T1106</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm x 12mm petri dishes</td>
			<td>Denville</td>
			<td>T1103</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>VWR</td>
			<td>300-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro dissection scissors</td>
			<td>Ted Pella</td>
			<td>1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting&#160;scissors, sharp tip, 41/2&#34;</td>
			<td>VWR</td>
			<td>82027-578</td>
			<td></td>
		</tr>
		<tr>
			<td>Watch glass square 1 5/8</td>
			<td>Carolina Biological Supply Company</td>
			<td>742300</td>
			<td>Autoclave before each use</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S8501</td>
			<td></td>
		</tr>
		<tr>
			<td>Trisodium Citrate Dihydrate</td>
			<td>Sigma</td>
			<td>S1804</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>646563</td>
			<td>Add to hypotonic buffer immediately before use</td>
		</tr>
		<tr>
			<td>50x Protease Inhibitor</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td>Add to hypotonic buffer immediately before use</td>
		</tr>
		<tr>
			<td>Turberculin syringe with needle (1cc, 27 G x 1/2 in)</td>
			<td>Becton, Dickinson (BD)</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold seal ultra frost glass slides (25X75mm, 1mm thick)</td>
			<td>Thermo</td>
			<td>3063-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Super PAP pen, large (liquid blocker)</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>71310</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde aqueous</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td>Detergent in manuscript</td>
		</tr>
		<tr>
			<td>Immuno stain moisture chamber</td>
			<td>Evergreen</td>
			<td>240-9020-Z10</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin jars</td>
			<td>Fisher</td>
			<td>08-815</td>
			<td></td>
		</tr>
		<tr>
			<td>Photo-flo 200</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>74257</td>
			<td>Wetting agent in manuscript</td>
		</tr>
		<tr>
			<td>Pregnant mare serum gonadotropin (PMSG)</td>
			<td>Sigma</td>
			<td>G4877</td>
			<td>Store aliquots at -20C</td>
		</tr>
		<tr>
			<td>Human chorionic gonadotropin (HCG)</td>
			<td>Sigma</td>
			<td>C0434</td>
			<td>Store aliquots at -20C</td>
		</tr>
		<tr>
			<td>PYREX Spot Plates with nine concave cavities</td>
			<td>Fisher</td>
			<td>13-748B</td>
			<td>Autoclave before each use</td>
		</tr>
		<tr>
			<td>Glass capillary</td>
			<td>Fisher</td>
			<td>22260943</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Brand Parafilm M</td>
			<td>Sigma</td>
			<td>BR701501</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Latex rubber tubing (3.2 mm inner diameter x 6.4 mm outer diameter)</td>
			<td>Fisher</td>
			<td>14-178-5B</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Latex rubber tubing (6.4 mm inner diameter x 11.1 mm outer diameter)</td>
			<td>Fisher</td>
			<td>14-178-5D</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Flexible silicone rubber nosepiece, hard plastic mouthpiece and 15 inches of latex tubing</td>
			<td>Sigma</td>
			<td>A5177-5EA</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Mitutoyo micrometer head</td>
			<td>Mituoyo</td>
			<td>150-208</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Syringe 5 mL</td>
			<td>BD Biosciences</td>
			<td>309646</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Syringe filter 0.45 &#956;m filter</td>
			<td>Corning</td>
			<td>431220</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Glass Pasteur Pipette 5 3/4&#34;</td>
			<td>Fisher</td>
			<td>13-678-6A</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>83 x 0.5 mm pipette tip</td>
			<td>Denville</td>
			<td>P3080</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Invitrogen</td>
			<td>11415049</td>
			<td></td>
		</tr>
		<tr>
			<td>Waymouth&#39;s media</td>
			<td>Life Technologies</td>
			<td>11220035</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Fisher</td>
			<td>A3160602</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A3311</td>
			<td>For oocyte culture media</td>
		</tr>
		<tr>
			<td>500ml filter units 0.22um</td>
			<td>Denville</td>
			<td>F5227</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrode&#8217;s solution</td>
			<td>Sigma</td>
			<td>T1788</td>
			<td>Store aliquots at -20C</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Sigma</td>
			<td>H-1270</td>
			<td>For ADB/wash buffer</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A1470</td>
			<td>For ADB/wash buffer</td>
		</tr>
		<tr>
			<td>VECTASHIELD antifade mounting medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover slides (22mmx60mm)</td>
			<td>Fisher</td>
			<td>12-544-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear nail polish</td>
			<td>Amazon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microscopes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SteREO Discovery.V8</td>
			<td>Zeiss</td>
			<td>495015-0001-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Observer Z1</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Primary Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-MLH1</td>
			<td>Life Technologies</td>
			<td>MA5-15431</td>
			<td>Dilution factor- 1:100</td>
		</tr>
		<tr>
			<td>Rabbit anti-SYCP1</td>
			<td>Life Technologies</td>
			<td>PA1-16763</td>
			<td>Dilution factor- 1:1000</td>
		</tr>
		<tr>
			<td>Goat anti-SCP3</td>
			<td>Santa Cruz</td>
			<td>sc-20845</td>
			<td>Dilution factor- 1:50</td>
		</tr>
		<tr>
			<td>Human anti-centromere protein</td>
			<td>Antibodies Incorporated</td>
			<td>15-235</td>
			<td>Dilution factor- 1:100</td>
		</tr>
		<tr>
			<td>Rabbit anti- TOPOII</td>
			<td>Abcam</td>
			<td>ab109524</td>
			<td>Dilution factor- 1:100</td>
		</tr>
		<tr>
			<td>Mouse anti-Histone H4 (di methyl K20, tri methyl K20)</td>
			<td>Abcam</td>
			<td>ab78517</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Rabbit anti-Rec8</td>
			<td>Courtesy of Dr. Karen Schindler</td>
			<td>N/A</td>
			<td>Dilution factor- 1:10,000</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Secondary Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-goat IgG (H+L) Alexa Fluor 568 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-11057</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse IgG (H+L) Alexa Fluor 488 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-21202</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit IgG (H+L) Alexa Fluor 647 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-31573</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (H+L) Alexa Fluor 568 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-11004</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-11013</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (H+L) Alexa Fluor 568 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-11011</td>
			<td>Dilution factor- 1:500</td>
		</tr>
	</tbody>
</table>

chromatin spread preparations for the analysis of mouse oocyte progression from prophase to metaphase ii

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

We have described two techniques for visualizing and assessing meiotic chromosomes in oocytes. The first technique is catered toward assessing prophase progression in embryonic and neonatal ovaries. Prophase chromatin spread preparations are incredibly valuable for visualizing numerous dynamic processes during meiosis, including chromosome pairing, synapsis and desynapsis, homologous recombination, and epigenetic chromosome remodeling. Here, we have demonstrated the utility of this method...

Watch this Scientific Journal Video about Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II at JoVE.com

Preparaty do rozprzestrzeniania się chromatyny do analizy progresji oocytów myszy od profazy do metafazy II | Genetyka | Wideo

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

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15.2K Views.  Johns Hopkins University Bloomberg School of Public Health. Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

Video: Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous nature of most mammalian chromatin-modifying complexes. In order to overcome these hurdles, we have adapted a sequential salt extraction (SSE) assay for evaluating the relative binding affinities of chromatin-bound complexes. This easy and straightforward assay can be used by non-experts to evaluate the relative difference in binding affinity of two related complexes, the changes in affinity of a complex when a subunit is lost or an individual domain is inactivated, and the change in binding affinity after alterations to the chromatin landscape. By sequentially re-suspending bulk chromatin in increasing amounts of salt, we are able to profile the elution of a particular protein from chromatin. Using these profiles, we are able to determine how alterations in a chromatin-modifying complex or alterations to the chromatin environment affect binding interactions. Coupling SSE with other in vitro and in vivo assays, we can determine the roles of individual domains and proteins on the functionality of a complex in a variety of chromatin environments.

Sequential salt extraction of chromatin bound proteins is a useful tool for determining the binding properties of large protein complexes. This method can be employed to evaluate the role of individual subunits or domains in the overall affinity of a protein complex to bulk chromatin.

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

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.

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

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

This work describes a protocol for chromatin immunoprecipitation (ChIP) using a mature mouse T-cell line. This protocol is suitable to investigate the distribution of specific histone marks at specific promoter sites or genome-wide.

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly useful. Hydrodynamic tail vein injection of transposon-based constructs is an efficient method for genetic manipulation of hepatocytes in adult mice. In addition to constitutive transgene expression, this system can be used for more advanced applications, such as shRNA-mediated gene knock-down, implication of the CRISPR/Cas9 system to induce gene mutations, or inducible systems. Here, the combination of constitutive CreER expression together with inducible expression of a transgene or miR-shRNA of choice is presented as an example of this technique. We cover the multi-step procedure starting from the preparation of sleeping beauty-transposon constructs, to the injection and treatment of mice, and the preparation of liver tissue for analysis by immunostaining. The system presented is a reliable and efficient approach to achieve complex genetic manipulations in hepatocytes. It is specifically useful in combination with Cre/loxP-based mouse strains and can be applied to a variety of models in the research of liver disease.

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Hydrodynamic tail vein injection of transposon-based integration vectors enables stable transfection of murine hepatocytes in vivo. Here, we present a practical protocol for transfection systems that enables the long-term constitutive expression of a single transgene or combined constitutive and doxycycline-inducible expression of a transgene or miR-shRNA in the liver.

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

This work describes a protocol for the generation of high resolution in situ Hi-C libraries from tightly staged pre-gastrulation Drosophila melanogaster embryos.

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...