Name: an integrated platform for genome-wide mapping of chromatin states using high-throughput chip-sequencing in tumor tissues
Uploaded: 2018-04-05T13:00:00
Description: Watch this Scientific Journal Video about An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues at JoVE.com

Vi takke Marcus Coyle, Curtis Gumbs, SMF kerne på MDACC for sekventering støtte. Det arbejde, der er beskrevet i denne artikel blev støttet af tilskud fra NIH grant (CA016672) til SMF kerne og NCI awards (1K99CA160578 og R00CA160578) til K. R.

Alle kliniske prøver blev opnået efter retningslinjerne i institutionelle Review Board.
1. buffer forberedelse
<ol><li>Gøre 200 mL af TE buffer (10 mM Tris-HCl, 10 mM ethylendiamintetra syre (EDTA) pH 8.0).</li>
	<li>Gøre 200 mL af STE buffer (10 mM Tris-HCl, 10 mM EDTA pH 8,0, 140 mM NaCl).</li>
	<li>Gøre 200 mL 2,0 M Glycin (37.52 g af glycin i 200 mL vand) og varme det til 65 ° C.</li>
	<li>Gøre 200 mL 5% natrium deoxycholate (DOC) opløsning (10 g af DOC i 200 mL vand).</li>
	<li>...

<ol>
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Denne protokol beskriver en komplet og omfattende høj overførselshastighed ChIP-seq modul for genome-wide kortlægning af kromatin stater i menneskelige tumor væv og cellelinjer. I ethvert ChIP-seq protokol er et af de vigtigste skridt antistof specificitet. Her, illustreres denne metode immunoprecipitation betingelser for de beskrevne seks Histon ændringer, som alle er ChIP-grade og tidligere har valideret i vores og andre laboratorier42,44,<sup c...

Fleste pattedyr genomer (98-99%) består af noncoding sekvens, og disse nocoding regioner indeholder regulerende elementer kendt for at deltage i kontrol af genekspression og kromatin organisation1,2. I en normal celle er den særlige samling af genomisk DNA i komprimerede kromatin struktur afgørende for den rumlige organisation, regulering og præcise timing af forskellige DNA-associerede processer3,4,5. I en kræftcelle dog kan kromatin ændringer af afvigende epigenetiske mekanismer føre til uretmæssig organisation af kromatin struktur, herunder adgang til regulerende elementer, kromosomale looping systemer og gen expression mønstre6, 7,8,9,10.
Trods de seneste fremskridt, har vi begrænset forståelse af epigenetiske ændringer, der er forbundet med tumor progression eller terapeutisk respons. Epigenome består af en række ændringer, herunder Histon mærker og DNA methylering, som samlet udgør et dynamisk tilstand (benævnt kromatin stat), der indvirker på gen expression netværk og andre processer, der er afgørende for at opretholde cellulære identitet. For nylig, ændringer i smagsforstærkere har været vist i flere maligniteter ved at studere H3K27Ac profiler11. Selv om sådanne undersøgelser giver indsigt i sammenhængen af isolerede epigenetiske mærker, mere end 100 epigenetiske ændringer har været identificeret12,13 uden klar forståelse af deres biologiske roller og indbyrdes afhængighed. Derudover er der et endnu større antal mulige kombinatorisk mønstre af disse Histon og DNA ændringer, og det er disse kombinatorisk mønstre - ikke enkelte ændringer - at dikterer epigenetiske stater14. Der er derfor enormt behov for at identificere ændringer i disse kromatin stater under kræft progression eller svar til terapi. Omfattende viden om epigenome forandringer i kræft har halter delvis på grund af tekniske (f.eks. generation af omfattende data fra små beløb af klinisk materiale/enkelt celler) og analytisk (fx algoritmer til at definere Kombinatorisk stater) udfordringer. Derfor er der kritiske behov for robust høj overførselshastighed metoder til profilering stort antal Histon ændring mærker fra klinisk materiale og let at implementere beregningsmæssige metoder til at forudsige kombinatorisk mønstre, som vil lette bestemmelse af epigenetiske stater forbundet med forskellige stadier af tumordannelse og terapeutiske modstand. Yderligere, data fra de seneste epigenome profilering undersøgelser15,16,17,18,19,20,21, 22,23 i normale væv og cellelinjer kan integreres med kromatin profiler af tumorer for yderligere indsigt i epigenome bidrag til tumor biology.
Kromatin profilering er blevet et stærkt værktøj til at identificere de globale bindende mønstre af forskellige kromatin ændringer15,24. I de seneste år blevet ChIP-FF. den "gyldne standard" for at studere DNA-protein interaktioner på en global skala25,26,27. Til ethvert ChIP-seq eksperiment er der kritiske trin nødvendigt for dens succes, herunder væv forarbejdning og afstandtagen, indhenting optimal sonikering betingelser, indhenting optimal antistof koncentration for nedbør, bibliotek forberedelse, efter sekvensering databehandling og downstream analyse. Hvert af disse trin indeholder centrale kvalitetskontrol checkpoints, og når det tages sammen, er afgørende for korrekt at identificere potentielle mål for funktionelle validering. Gennem innovation i disse trin, har flere forudgående undersøgelser udviklet metoder til at udføre ChIP eller ChIP-Seq fra lille mængde væv28,29,30,31,32 . Yderligere, nogle undersøgelser har antydet, protokoller for høj overførselshastighed ChIP eksperimenter efterfulgt af PCR baseret kvantitering33,34. Endelig er nogle offentligt tilgængelige analyse platforme for ChIP-Seq data nu tilgængelige som Easeq35 og Galaxy36. Dog har en integreret platform til at udføre ChIP-Seq i en høj overførselshastighed mode i kombination med en beregningsmæssige rørledning til udføre enkelt mark samt kromatin stat analyser manglet.
Denne protokol beskriver en komplet og omfattende ChIP-seq arbejdsproces for genome-wide kortlægning af kromatin stater i tumor væv og cellelinjer, med let for at følge retningslinjerne omfatter alle de trin, der er nødvendige for en vellykket eksperiment. Ved at vedtage en høj overførselshastighed, der er tidligere beskrevet af Blecher-Gonen et al. 37, denne protokol kan udføres på snesevis af prøver i parallel og er blevet anvendt på kræft cellelinjer og menneskelige tumorer såsom melanom, tyktarmen, prostata og glioblastom multiforme. Vi demonstrere metodologien for seks core Histon modifikationer, som udgør centrale elementer i den epigenetiske lovgivningslandskab i menneskelige melanom cellelinjer og tumor prøver. Disse ændringer omfatter H3K27ac (smagsforstærkere), H3K4me1 (aktiv og klar smagsforstærkere), H3K4me3 (initiativtagere), H3K79me2 (transskriberet regioner), H3K27me3 (polycomb undertrykkelse), og H3K9me3 (heterochromatic undertrykkelse). Disse mærker kan bruges enten alene eller i kombination til at identificere funktionelt adskilte kromatin stater repræsenterer både repressive og aktive domæner.

<table>
	<tbody>
		<tr>
			<td>ChIP-grade H3K4me1 antibody</td>
			<td>Abcam</td>
			<td>ab8895</td>
			<td></td>
		</tr>
		<tr>
			<td>ChIP-grade H3K27ac antibody</td>
			<td>Abcam</td>
			<td>ab4729</td>
			<td></td>
		</tr>
		<tr>
			<td>ChIP-grade H3K4me3 antibody</td>
			<td>Abcam</td>
			<td>ab8580</td>
			<td></td>
		</tr>
		<tr>
			<td>ChIP-grade H3K79me2 antibody</td>
			<td>Abcam</td>
			<td>ab3594</td>
			<td></td>
		</tr>
		<tr>
			<td>ChIP-grade H3K27me3 antibody</td>
			<td>Abcam</td>
			<td>ab6002</td>
			<td></td>
		</tr>
		<tr>
			<td>ChIP-grade H3K9me3 antibody</td>
			<td>Abcam</td>
			<td>ab8898</td>
			<td></td>
		</tr>
		<tr>
			<td>1M Tris HCl, pH 8.0</td>
			<td>Teknova</td>
			<td>T1080</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>30970</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Sigma - Life Sciences</td>
			<td>D8537-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich</td>
			<td>74255</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma-Aldrich</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>LiCl</td>
			<td>Sigma-Aldrich</td>
			<td>746460</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Calbiochem</td>
			<td>492016-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>1% TWEEN-20</td>
			<td>Fisher Bioreagents</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA - IgG-free</td>
			<td>Sigma - Life Sciences</td>
			<td>A2058-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibo</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>GentleMACS C tube</td>
			<td>GentleMACS</td>
			<td>120-008-466</td>
			<td>disassociation tube</td>
		</tr>
		<tr>
			<td>16% Formaldehyde</td>
			<td>Peierce</td>
			<td>28906</td>
			<td></td>
		</tr>
		<tr>
			<td>miniProtease inhibitor&#160;</td>
			<td>Roche Diagnostics</td>
			<td>11836153001</td>
			<td>protease inhibitor tablets</td>
		</tr>
		<tr>
			<td>Dynabeads Protein G&#160;</td>
			<td>Invitrogen</td>
			<td>10009D</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioruptor NGS tubes 0.65 mL</td>
			<td>Diagenode</td>
			<td>C30010011</td>
			<td>sonication tubes</td>
		</tr>
		<tr>
			<td>DynaMag - 96 Side Skirted</td>
			<td>Invitrogen</td>
			<td>120.27</td>
			<td>96-well magnetic stand</td>
		</tr>
		<tr>
			<td>TE buffer</td>
			<td>Promega</td>
			<td>V6231</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>Invitrogen</td>
			<td>12091021</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Invitrogen</td>
			<td>100005393</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPure XP beads</td>
			<td>Beckman Coulter</td>
			<td>A63882</td>
			<td>paramagnetic beads</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit ds DNA High Sensitivity Assay Kit</td>
			<td>Invitrogen</td>
			<td>Q32854</td>
			<td>high sensitivity DNA reagents</td>
		</tr>
		<tr>
			<td>NEBNext Ultra II DNA Library Prep Kit</td>
			<td>New England BioLabs</td>
			<td>E7645L</td>
			<td>DNA Library Prep Kit</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Ambion</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>High sensitivity D1000 ScreenTape</td>
			<td>Agilent Technologies</td>
			<td>5067-5584</td>
			<td>high sensitivity DNA reagents</td>
		</tr>
		<tr>
			<td>High sensitivity D1000 reagents</td>
			<td>Agilent Technologies</td>
			<td>5067-5585</td>
			<td>high sensitivity DNA reagents</td>
		</tr>
		<tr>
			<td>Multiplex Oligos (Index primers- Set 1)</td>
			<td>New England BioLabs</td>
			<td>E7335L</td>
			<td>Multiplex Oligos&#160;</td>
		</tr>
		<tr>
			<td>Multiplex Oligos (Index primers- Set 2)</td>
			<td>New England BioLabs</td>
			<td>E7500L</td>
			<td>Multiplex Oligos&#160;</td>
		</tr>
		<tr>
			<td>TapeStation 4200</td>
			<td>Agilent Technologies</td>
			<td>G2991AA</td>
			<td>high sensitivity DNA electropherogram instrument</td>
		</tr>
		<tr>
			<td>Bioruptor Pico sonication device&#160;</td>
			<td>Diagenode</td>
			<td>B01060001</td>
			<td>water bath disruputor</td>
		</tr>
		<tr>
			<td>Mixer</td>
			<td>Nutator</td>
			<td>421105</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad C1000 Touch Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td>1851196</td>
			<td>PCR Thermal cycler</td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>Fisher Scientific</td>
			<td>2322</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel Pipet</td>
			<td>Denville</td>
			<td>1003123</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube Revolver</td>
			<td>Thermo-Scientific</td>
			<td>88881001</td>
			<td></td>
		</tr>
		<tr>
			<td>96-Well Skirted Plate</td>
			<td>Eppendorf</td>
			<td>47744-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra X-12R Centrifuge&#160;</td>
			<td>Beckman Coulter</td>
			<td>A99464</td>
			<td>benchtop centrifuge</td>
		</tr>
		<tr>
			<td>Centrifuge 5424</td>
			<td>Eppendorf</td>
			<td>22620461</td>
			<td>tabletop centrifuge</td>
		</tr>
		<tr>
			<td>Optical tube strips (8x Strip)</td>
			<td>Agilent Technologies</td>
			<td>401428</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical tube strip caps (8x strip)</td>
			<td>Agilent Technologies</td>
			<td>401425</td>
			<td></td>
		</tr>
		<tr>
			<td>Loading Tips, 10 Pk</td>
			<td>Agilent Technologies</td>
			<td>5067-5599</td>
			<td></td>
		</tr>
		<tr>
			<td>IKA MS3 vortex</td>
			<td>IKA</td>
			<td>3617000</td>
			<td>vortex</td>
		</tr>
	</tbody>
</table>

an integrated platform for genome-wide mapping of chromatin states using high-throughput chip-sequencing in tumor tissues

Histon ændringer udgør en større komponent af epigenome og spiller en vigtig lovgivningsmæssig rolle ved fastsættelsen af transcriptional status af associerede loci. Derudover er tilstedeværelsen af specifikke ændringer blevet brugt til at bestemme den holdning og identitet ikke-kodende funktionelle elementer såsom smagsforstærkere. I de seneste år, er kromatin immunoprecipitation efterfulgt af næste generation sequencing (ChIP-FF.) blevet et stærkt værktøj til at bestemme genome-wide profiler af individuelle Histon ændringer. Dog er det blevet stadig tydeligere, at de kombinatorisk mønstre af kromatin ændringer, omtales som kromatin stater, bestemme identitet og karakter af den tilknyttede genomisk locus. Derfor arbejdsprocesser bestående af robust høj overførselshastighed (HT) metoder til profilering en række Histon ændring mærker, samt beregningsmæssige analyser rørledninger i stand til at håndtere myriader af ChIP-Seq profilering datasæt, er nødvendige for omfattende bestemmelse af epigenomiske stater i stort antal prøver. The HT-ChIP-Seq arbejdsproces præsenteres her består af to moduler: 1) en forsøgsplan for profilering flere Histon ændringer fra små mængder af tumor prøver og cellelinjer i 96-brønds format. og 2) et beregningsmæssige data analyse rørledning, der kombinerer eksisterende værktøjer til at beregne både individuelle mark belægning og kombinatorisk kromatin tilstand mønstre. Sammen, lette disse to moduler nem behandling af hundredvis af ChIP-Seq prøver på en hurtig og effektiv måde. Arbejdsprocessen præsenteres her bruges til at udlede kromatin tilstand mønstre fra 6 Histon mark profiler i melanom tumorer og cellelinjer. Samlet, præsenterer vi en omfattende ChIP-seq arbejdsproces, der kan anvendes til snesevis af menneskelige tumor prøver og kræft cellelinjer til at bestemme epigenomiske aberrationer i forskellige maligniteter.

Denne protokol tillader immunoprecipitation fra frosne tumor væv og cellelinjer, der kan udføres på snesevis af prøver parallelt med en høj overførselshastighed metode (figur 1A). Kromatin fragmenter bør variere mellem ~ 200-1000 bps for optimal immunoprecipitation. Vi har bemærket, at tid til at opnå samme klipning længde er forskellig for forskellige væv og celletyper. ChIP fra små mængder af væv succes afhænger holde lysate ...

Watch this Scientific Journal Video about An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues at JoVE.com

An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues

En integreret Platform for Genome-wide kortlægning af kromatin stater ved hjælp af høj overførselshastighed ChIP-sekventering i Tumor væv

Her, beskriver vi en optimeret høj overførselshastighed ChIP-sekventering protokol og beregningsmæssige analyser rørledning til bestemmelse af genome-wide kromatin tilstand mønstre fra frosne tumor væv og cellelinjer.

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of ...

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