Name: chromatin immunoprecipitation (chip) of histone modifications from saccharomyces cerevisiae
Uploaded: 2017-12-29T15:00:00
Description: Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae at JoVE.com

Författarna vill tacka medlemmarna i gröna lab för bra diskussioner. Detta arbete stöds delvis av NIH grants R03AG052018 och R01GM124342 till E.M.G.

1. före binda antikroppar mot magnetiska pärlor
<ol>
	<li>Blanda protein A/G magnetiska pärlor väl och överföra 20 µL av magnetiska pärlor per en immunoprecipitation (IP) prov till en 1,5 mL tub. Obs: När pipettering pärlor, använda wide-bore, låg-lagring tips.</li>
	<li>Ställ röret med pärlor i en magnetisk stå och pärlor för att samla på sidan av röret. Ta bort supernatanten.</li>
	<li>Tvätta pärlor 3 gånger med 1 mL kall Tris-buffrad koksaltlösning (TBS) (50 mM Tris-HCl pH 7.5, 150 mM NaCl...

<ol>
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Proceduren som beskrivs här möjliggör effektiv återvinning av DNA som är associerad med modifierade histoner i jästceller av immunoprecipitation. Detta följs av qPCR med primers som förstärker regioner av intresse att bestämma lokala anrikning eller utarmning av specifika Histon ändringar. Trots att utvecklas som en metod för nästan 20 år sedan, fortfarande chIP definierande analysen för att utreda Histon ändring status på olika genomisk regioner och under olika förhållanden. Även om chIP kopplat till...

Dynamiska posttranslationella modifieringen (PTM) av histoner är en viktig reglerande mekanism för många DNA-mallade processer, inklusive transkription, replikering och DNA reparation1,2. Möjligheten att bestämma överflöd och exakt lokalisering av modifierade histoner samtidig med dessa processer är därför avgörande för att förstå deras reglering under olika förhållanden i cellen. Utvecklingen av kromatin immunoprecipitation (chIP) som en metod som till stor del härrörde från biokemiska studier av interaktioner mellan proteiner och DNA, bestämt in vitro- metoder med kemiska bindemedel, tillsammans med behovet av att utvärdera den dynamiska karaktär av protein-DNA interaktioner i vivo och på specifika regioner av genomet3,4,5. Befordran av kvantitativ PCR (qPCR) och sekvensering teknik har också utökat möjligheten att utföra chIP experiment med kvantitativa jämförelser och över hela genomet, vilket gör det ett kraftfullt verktyg för dissekera DNA-protein interaktioner på flera nivåer.
För närvarande är chIP en lämplig metod för alla intresserade av kromatin-medierad reglering av genomet forskargrupp som det finns inga jämförbara metoder för direkt förhör den fysiska kopplingen mellan en modifierad Histon och en specifik genomisk locus i vivo. Även om det finns varianter av denna metod som använder nästa generations sekvensering mappa Histon ändringar i hela genomet6,7 , kan dessa synsätt behandla olika vetenskapliga frågor och deras skala, kostnad och tekniska resurser kan vara begränsande för vissa forskargrupper. Dessutom riktade chIP-qPCR är nödvändigt att komplettera dessa metoder genom att tillhandahålla metoder både optimera chIP protokollet före sekvensering och att validera resultaten från epigenetisk datamängderna. Masspektrometri baserad metoder för att identifiera full uppsättning av Histon märken förknippas med genomiska regioner har också dykt upp8,9,10,11, dock dessa metoder har vissa begränsningar angående vilka regioner i genomet kan vara utforskad och de kräver teknisk expertis och instrumentering som inte kommer att vara tillgängliga för alla forskargrupper. ChIP förblir därför en grundläggande metod för att analysera överflöd och distribution av Histon ändringar under varierande förhållanden för alla forskargrupper intresserad epigenetik, kromatin och regleringen av genomisk funktioner.
Här, beskriver vi en metod för chIP med spirande jästen modell Saccharomyces cerevisiae (S. cerevisiae) för att undersöka fördelningen av Histon PTMs på kromatin. Denna strategi bygger på ett antal centrala delarna av chIP protokoll utvecklad i jäst och också tillämpas på olika modell system12,13. Interaktioner mellan modifierade histoner och DNA i cellen bevarade av crosslinking med formaldehyd. Efter lysate beredning, kromatin fragment är solubilized i enhetligt medelstora fragment av matsmältning med micrococcal nuclease. Immunoprecipitation av modifierade Histonerna utförs med antingen kommersiella eller lab-genererade antikroppar och eventuella associerade DNA är isolerade och analyseras för berikning på särskilda genomisk regioner med qPCR (figur 1). För många Histon modifieringar räcker mängden DNA som erhållits från detta protokoll för att testa mer än 25 olika genomisk loci av qPCR.
Detta chIP metod är mångsidig för att övervaka distribution av en enstaka Histon ändring på flera mutant stammar eller miljöförhållanden, eller för att testa flera Histon ändringar i vildtyp celler på ett antal genetiska loci. Dessutom är många komponenter i protokollet enkelt justerbar att optimera upptäckt av antingen högt - eller ödmjuk-riklig Histon-märken. Slutligen ger utför chIP av modifierade histoner i spirande jäst möjlighet att använda viktiga kontroller för antikropp specificitet som till stor del inte är tillgängliga i andra system. Nämligen jäststammar kan genereras som bär punktmutationer i Histon rester som är avsedda för modifiering, och, i vissa fall finns det bara ett enda enzym som katalyserar ändring på en viss Histon rester (t.ex. Histon lysin metyltransferaser). Därför kan chIP utföras i antingen Histon mutant eller enzym radering stammar till assay i omfattning som icke-specifik bindning av antikroppen kan förekommande och generera falskt positiva resultat. Denna kontroll är särskilt värdefulla för nyutvecklade antikroppar, och kan även användas för att validera antikropp specificitet för bevarade Histon ändringar före deras användning i andra system. Detta synsätt kompletterar andra metoder för att testa antikropp specificitet som skiljer mellan olika modifiering stater (t.ex. mono-, di- och tri-metylering), inklusive sondera matriser av modifierade peptider och utföra western blotting av histoner eller nucleosomes med definierade ändringar. Övergripande, chIP i spirande jäst är en kraftfull metod för att bedöma dynamiken i Histon PTMs i hela genomet och dissekera de mekanismer som styr deras reglering.

<table border="0" cellpadding="0" cellspacing="0" width="876">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Yeast Extract</td>
			<td style="width:301px;">Research Products International (RPI)</td>
			<td style="width:164px;">Y20025-1000.0</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Peptone</td>
			<td style="width:301px;">Research Products International (RPI)</td>
			<td style="width:164px;">P20250-1000.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Dextrose</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">BP350-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Glycine</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td>AC12007-0050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Tris</td>
			<td style="width:301px;">Amresco</td>
			<td style="width:164px;">0497-5KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E6758-500G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">NaCl</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">BP358-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Nonylphenyl-polyethylene glycol</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">74385</td>
			<td>Equivalent to NP-40</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">MgCl</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">S25533</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:244px;">CaCl2</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">20899-25G-F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">LiCl</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">AC413271000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Sodium Dodecyl Sulfate</td>
			<td style="width:301px;">Amresco</td>
			<td style="width:164px;">M107-1KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Sodium Deoxycholate</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">30970-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Sodium Acetate</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">S2889</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:244px;">NaHCO3</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">S25533</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>P7626-5G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Yeast protease inhibitor cocktail</td>
			<td>VWR</td>
			<td>10190-076</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">25 Phenol:24 Chloroform:1 Isoamyl Alcohol</td>
			<td style="width:301px;">VWR Life Science</td>
			<td>97064-824</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Ethanol</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">E7023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Nuclease-Free Water</td>
			<td style="width:301px;">VWR</td>
			<td style="width:164px;">100720-992</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Micrococcal Nuclease</td>
			<td>Worthington Biochemical</td>
			<td>LS004797</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Glycogen</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">R0561</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Proteinase K</td>
			<td>Research Products International (RPI)</td>
			<td>P50220-0.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">RNase A&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>R6513-50MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;Bradford Assay Reagent</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">23238</td>
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			<td height="20" style="height:20px;width:244px;">&#160;BSA Standard 2 mg/mL</td>
			<td style="width:301px;">ThermoScientific</td>
			<td>23210</td>
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			<td height="20" style="height:20px;width:244px;">&#945; H4</td>
			<td>EMD Millipore</td>
			<td>04-858</td>
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			<td height="20" style="height:20px;width:244px;">&#945; H4K16ac</td>
			<td>EMD Millipore</td>
			<td>ABE532</td>
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			<td height="20" style="height:20px;width:244px;">&#945; H3</td>
			<td style="width:301px;">Abcam</td>
			<td style="width:164px;">ab1791</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H3K4me2</td>
			<td>Active Motif</td>
			<td>39142</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;High Rox qPCR Mix</td>
			<td>Accuris qMax Green, Low Rox qPCR Mix</td>
			<td>ACC-PR2000-L-1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Protein A/G Magnetic Beads</td>
			<td style="width:301px;">ThermoScientific</td>
			<td>88803</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">magnetic stand for 1.5mL tubes</td>
			<td>Fisher Scientific</td>
			<td>PI-21359</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Acid-Washed Glass Beads</td>
			<td>Sigma-Aldrich</td>
			<td style="width:164px;">G8772</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;Microtube Homogenizer</td>
			<td style="width:301px;">Benchmark</td>
			<td>D1030</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:244px;">2.0 mL screw-cap tubes with sealing rings</td>
			<td>Sigma-Aldrich</td>
			<td>Z763837-1000EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Gel loading tips</td>
			<td>Fisher Scientific</td>
			<td>07-200-288</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Cuvettes</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td style="width:164px;">50-476-476</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Parafilm</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td>13-374-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">50 mL conical tubes</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td style="width:164px;">14-432-22</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">384-Well PCR Plate</td>
			<td>Fisher Scientific</td>
			<td>AB-1384W</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;Gyratory Floor Shaker</td>
			<td style="width:301px;">New Brunswick Scientific</td>
			<td style="width:164px;">Model G10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Spectrophotometer</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">ND-2000c</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">&#160;Real-Time PCR Detection System</td>
			<td style="width:301px;">Bio-Rad</td>
			<td style="width:164px;">1855485</td>
			<td></td>
		</tr>
	</tbody>
</table>

chromatin immunoprecipitation (chip) of histone modifications from saccharomyces cerevisiae

Histon post-translationella modifieringar (PTMs), såsom acetylering, metylering och fosforylering, regleras dynamiskt av en rad enzymer som Lägg till eller ta bort dessa märken som svar på signaler tas emot av cellen. Dessa PTMS är nyckelpersoner till regleringen av processer såsom gen uttryck kontroll och DNA reparation. Kromatin immunoprecipitation (chIP) har varit en instrumental tillvägagångssätt för dissekera överflöd och lokalisering av många Histon PTMs i hela genomet som svar på olika störningar till cellen. Här beskrivs en mångsidig metod för att utföra post-translationally modifierade histoner-chIP från spirande jästen Saccharomyces cerevisiae (S. cerevisiae) . Denna metod förlitar sig på crosslinking av proteiner och DNA använder formaldehyd behandling av jäst kulturer, generation av jäst lysates av pärla slå, lösbarhet av kromatin fragment av micrococcal nuclease och immunoprecipitation Histon-DNA komplex. DNA är associerad med varumärket Histon sevärdheter renas och föremål för kvantitativa PCR-analys att utvärdera dess anrikning på flera loci i hela genomet. Representativa experiment sondera localizationen av Histon varumärkena H3K4me2 och H4K16ac i vildtyp och muterade jäst diskuteras för att Visa dataanalys och tolkning. Denna metod är lämplig för en mängd Histon PTMs och kan utföras med olika muterade stammar eller i närvaro av olika miljöpåfrestningar, vilket gör det till ett utmärkt verktyg för att undersöka förändringar i kromatin dynamik under olika förhållanden.

En viktig komponent i detta protokoll är att optimera koncentrationen av micrococcal nuclease (MNase) används för att smälta kromatinet i lösliga fragment, som beskrivs i steg 10. Detta är avgörande för att erhålla hög upplösning data angående distributionen av Histon ombyggnadsar genomisk regioner av intresse. En MNase titrering bör utföras för att bestämma den mest lämpliga koncentrationen att uppnå primärt mono-nucleosomes med en mindre mängd di-nucleosomes i lösli...

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae at JoVE.com

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Kromatin Immunoprecipitation (ChIP) Histon ändringar från Saccharomyces cerevisiae

Här beskriver vi ett protokoll för kromatin immunoprecipitation av modifierade histoner från spirande jästen Saccharomyces cerevisiae. Immunoprecipitated DNA används därefter för kvantitativ PCR för att förhöra överflöd och lokalisering av Histon post-translationella modifieringar i hela genomet.

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

The overall goal of this chromatin immunoprecipation protocol is to determine the abundance and localization of histone modifications at defined regions throughout the budding yeast genome. This method can help answer key questions in chromatin regulation, such as how histone modifications are linked to gene expression control and how these modifications change in response to different environmental conditions. An extremely versatile protocol that can be used to investigate many different histone modifications and multiple E strains at one time.Though this method can provide insight into histone modifications, it can also be applied to investigating other chromatin bonding proteins through the use of epitope tags or available antibodies. Start this procedure by growing the yeast cells inoculate 10 milliliters of YPD medium with a single colony of each appropriate strain, and grow it 30 degrees celsius in a shaking incubator at 220 rpm overnight. On the following day after measuring the optical density at 600 nanometers or OD600 of the culture, dilute the culture to an OD600 of 0.2 in 100 mL of YPD in a new flask.Grow the cells at 30 degree celsius in a shaking incubator at 220 rpm until they reach mid log phase. When the cultures have reached mid log phase, add 2.7 milliliters of 37%formaldehyde directly to the medium in each flask for a final concentration of 1%formaldehyde. Transfer the flasks to a shaker at room temperature, and shake at 50 rpm for 15 minutes.Add five milliliters of 2.5 molar glycine to the medium and continue shaking at 50 rpm at room temperature for five minutes to quench the formaldehyde. Subsequently, spin down the cells and wash them as described in the text protocol. Remove the supernatant after the last spin and proceed to making yeast lysates.Re-suspend the cells in one milliliter of cold chromatin amino precipitation or ChIP Lysis buffer with 1 millimolar PMSF and 1:1000 dilution of a yeast protease inhibitor cocktail. Transfer the suspension to a 2.0 milliliter screw cap tube containing 200 microliters of glass beads. Transfer the cells to a bead-beating apparatus for a high speed agitation at four degree celsius.Bead-beat for 30 seconds and ice for one minute. Bead-beat a total of six times, keeping the cells on ice for at least one minute between each 30 second beat-beating. Using a gel loading tip, transfer the lysate to a 1.5 milliliter tube.Centrifuge the lysate at 15, 500 g at four degree celsius for 20 minutes. Discard the supernatant and re-suspend the pellet in 250 microliters of micrococcal nuclease digestion buffer by gently pipetting up and down. Add 2.5 microliters of micrococcal nuclease to each reaction.Mix gently by inverting four to six times and immediately incubate in a 37 degree celsius water bath for 20 minutes. After 20 minutes, stop the reaction by placing the tubes on ice, adding five milliliters of 0.5 molar EDTA for a final concentration of 10 millimolar EDTA and mixing gently by inversion. Centrifuge at 15, 500 g at four degree celsius for 15 minutes.Then, transfer each supernatant to a new 1.5 milliliter tube. For the most consistent results, it is critical to ensure that the same concentration of protein is used in each amino precipitation. Depending on the number of amino precipitation or IP reactions, make a master mix of each micrococcal nuclease released chromatin fraction and ChIP lysis buffer and allocate one milliliter containing 50 micrograms of total protein to individual tubes.Remove 100 microliters from each IP mix to process as the input sample. Freeze the input samples at 20 degree celsius for later use. It is also important to make sure the same amount of beads bound to the antibody is used for each immunoprecipitation.Using a wide board pipette tip, add 20 microliters of magnetic protein AG beads, pre-bound with antibody to each IP sample. Rotate the samples at eight rpm at four degree celsius for three hours. When the IP is done, place the tubes in a magnetic stand and let beads collect on the side of the tube.Use a pipette to remove the supernatant. Add one milliliter of ChIP lysis buffer to the beads and rotate at eight rpm at four degree celsius for five minutes. Then, place the tubes on the magnetic stand and remove the supernatant.In this manner, wash the beads successively with the following buffers for the number of times indicated. ChIP Lysis Buffer, High Salt Wash Buffer, LiCl/detergent Wash Buffer and TE.With the last TE wash, use 0.5 milliliters of TE to transfer most of the beads to a new tube, and then use another 0.5 milliliters of TE to wash the tube and transfer the remaining beads. Add 250 microliters of freshly made ChIP elution buffer to each tube and vortex the solution briefly.Rotate the samples at eight rpm at room temperature for 15 minutes. Place the tubes in the magnetic stand. Carefully transfer the supernatant to a new tube and avoid the beads.Add another 250 microliters of ChIP elution buffer to the beads. Rotate the samples at eight rpm at room temperature for 15 minutes. Carefully and without disturbing the beads, transfer the supernatant to the tube containing the first elution.Thaw the input samples collected earlier and add 400 microliters of ChIP elution buffer to each input sample. To reverse protein DNA cross links, add to both input and IP samples, 20 microliters of five molar sodium chloride, five microliters of glycogen and 12.5 microliters of Proteinase K.Mix well by inverting or flicking the tubes. Incubate the samples in a 65 degree celsius water bath overnight.To begin this procedure, make a QPCR master mix for each set of primers by combining the appropriate volumes of QPCR mix, the forward primer, and the reverse primer. Make DNA master mixes for each DNA sample where one reaction contains 0.5 microliters of DNA and 4.0 microliters of nuclease free water Add 5.5 microliters of the appropriate primer master mix to each well in a 384 well QPCR plate. Spin the plate at 200 g at room temperature for one minute.Then, add 4.5 microliters of the appropriate DNA master mix to each well. Seal the plate, and spin at 200 g at room temperature for one minute. Perform QPCR using a real time QPCR system with the following conditions, 95 degree celsius for three minutes 40 cycles of 95 degree celsius for 10 seconds and 55 degree celsius for 30 seconds and a melting curve of 65 degree celsius to 95 degree celsius with 0.5 degree celsius increments for five seconds.One key component of this protocol is optimizing the concentration of micrococcal nuclease used to digest the chromatin into soluble fragments. This example result from agarose gel electrophoresis of DNA isolated following digestion of the chromatin pellet with varying amounts of micrococcal nuclease shows that 2.5 microliters of enzyme at 20 units per microliter produced predominantly mononucleosomes. This representative ChIP experiment used antibodies against a histone methyl marker H3K4me2 and histone H3 in wild type and set1 knockout cells.Set1 knockout cells lack the methyl transferase that catalyzes the mark The bars indicate the relative enrichment of H3K4me2 at the indicated low sci. The wild type strain showed clear enrichment for H3K4me2 at the five prime end of two genes known to be direct targets of set1, PMA1 and ERG11 whereas no signal was observed in set1 knockout cells. As expected, there was no enrichment of the H3K4me2 mark at TEL07L, the expression of SPR3 has also been reported to be regulated by set1 but these results indicate the regulation is likely to occur by a different mechanism than at PMA1 or ERG11.Once mastered, this technique can be done within four days if it is performed properly. While attempting this procedure, it's important to remember to carefully test the antibody used for specificity with the proper controls. If available, both positive and negative control regions should be probed in the QPCR.Following this procedure, other methods like GP sequencing can also be performed in order to determine the genome by distribution of histone modifications. After watching this video, you should have a good understanding of how to consistently detect the levels of different histone modifications at unique genomic regions with well controlled experiments.

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Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

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Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription ...