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 takker Marcus Coyle, Curtis Gumbs, SMF kjernen for MDACC for sekvensering støtte. Arbeidet som er beskrevet i denne artikkelen ble støttet av tilskudd fra NIH tilskudd (CA016672) til SMF kjerne og NCI priser (1K99CA160578 og R00CA160578) til K. R.

Alle kliniske prøvene ble innhentet etter retningslinjene for institusjonelle Review Board.
1. buffer forberedelse
<ol><li>Gjøre 200 mL TE buffer (10 mM Tris-HCl, 10 mM ethylenediaminetetraacetic syre (EDTA) pH 8.0).</li>
	<li>Gjøre 200 mL STE buffer (10 mM Tris-HCl, 10 mM EDTA pH 8.0, 140 mM NaCl).</li>
	<li>Gjøre 200 mL 2.0 M glysin (37.52 g av glysin i 200 mL vann) og varme den til 65 ° C.</li>
	<li>Gjøre 200 mL 5% natrium deoxycholate (DOC) løsning (10 g av DOC i 200 mL vann).</li...

<ol>
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Denne protokollen beskriver en komplett og omfattende høy gjennomstrømming ChIP-seq modul for genomet hele kartlegging av chromatin statene i menneske tumor vev og linjer. I noen ChIP-seq-protokollen er en av de viktigste trinnene antistoff spesifisitet. Her viser denne metoden immunoprecipitation betingelser for beskrevet seks histone modifikasjoner, alle er ChIP-klasse og tidligere har validert i våre og andre laboratorier42,44,4...

Fleste pattedyr genomer (98-99%) består av noncoding, og disse nocoding områdene inneholder regulatoriske elementer kjent å delta i å kontrollere genuttrykk og chromatin organisasjonen1,2. I en normal celle er bestemte forsamlingen genomisk DNA komprimerte chromatin strukturen avgjørende for romlig organisering, regulering og presis timing av ulike DNA-assosiert prosesser3,4,5. I en kreftcelle imidlertid kan chromatin modifikasjoner av avvikende epigenetic mekanismer føre til feil organisasjon av chromatin struktur, inkludert tilgang til regulatoriske elementer, chromosomal looping systemer og gene expression mønstre6, 7,8,9,10.
Til tross for nylige fremskritt, har vi begrenset forståelse av epigenetic endringer som er tilknyttet svulst progresjon eller terapeutisk svar. Epigenome består av en rekke endringer, inkludert histone merker og DNA metylering, som samlet utgjør en dynamisk tilstand (referert til som chromatin stat) som gjøre inngrep på genet uttrykk nettverk og andre prosesser som er avgjørende for å opprettholde mobilnettet identitet. Endringer i enhancers har nylig vist i flere malignitet ved å studere H3K27Ac profiler11. Selv om slike studier gir innsikt i sammenhengen isolert epigenetic merker, mer enn 100 epigenetic endringer har blitt identifisert12,13 uten klar forståelse av deres biologiske roller og avhengighet. Videre finnes det et enda større antall mulige kombinasjon mønstre av disse histone og DNA modifikasjoner, og det er disse kombinasjon mønstre - ikke endinger - dikterer epigenetic stater14. Derfor er det enormt behov å identifisere endringer i disse chromatin stater under kreft progresjon eller svar til terapi. Omfattende kunnskap om epigenome endringer i kreft er lagging delvis på grunn av tekniske (f.eks generasjon av store data fra liten mengde klinisk materiale/single celler) og analytisk (f.eks algoritmer til å definere kombinasjon stater) utfordringer. Derfor er det kritisk behov for robust høy gjennomstrømming metoder for profilering stort antall histone endring merker fra klinisk materiale og lett-å-implementere computational tilnærminger til å forutsi kombinasjon mønstre som vil lette fastsettelse av epigenetic knyttet til ulike stadier av tumorigenesis og terapeutiske motstand. Videre, data som er tilgjengelige fra siste epigenome profilering studier15,16,17,18,19,20,21, 22,23 i normalt vev og linjer kan integreres med chromatin profiler av svulster for ytterligere innsikt i epigenome bidrag til svulst biologi.
Chromatin profilering har blitt et kraftig verktøy for å identifisere globale bindende mønstre av ulike chromatin modifikasjoner15,24. De siste årene blitt ChIP-seq "gull standard" for å studere DNA-protein interaksjoner på en global skala25,26,27. For noen ChIP-seq eksperiment er det avgjørende skritt nødvendig for sin suksess, inkludert vev behandling og tilknytningene, finne optimale sonication forhold, finne optimale antistoff konsentrasjon for nedbør, bibliotek forberedelse, etter sekvensering databehandling, og nedstrøms. Hvert av disse trinnene inneholder viktige kvalitetskontroll sjekkpunkter, og samlet, er avgjørende for riktig identifisere potensielle mål for funksjonell validering. Gjennom innovasjon i disse trinnene, har flere tidligere studier utviklet metoder for å utføre ChIP eller ChIP-Seq fra liten mengde vev28,29,30,31,32 . Videre, noen studier har antydet protokoller for høy gjennomstrømming ChIP eksperimenter etterfulgt av PCR basert kvantifisering33,34. Endelig er noen offentlig tilgjengelig analyse plattformer for ChIP-Seq-data nå tilgjengelig som Easeq35 og Galaxy36. Imidlertid har en integrert plattform for å utføre ChIP-Seq i en høy gjennomstrømming mote i kombinasjon med en beregningsorientert rørledning utføre enkelt merke samt chromatin staten analyser manglet.
Denne protokollen beskriver en komplett og omfattende ChIP-seq arbeidsflyt for genomet hele kartlegging av chromatin stater i tumor vev og cellelinjer, med lett for å følge retningslinjene omfatter alle trinnene nødvendig for en vellykket eksperiment. Ved å vedta en høy gjennomstrømming metode tidligere beskrevet av Blecher-Gonen et al. 37, denne protokollen kan utføres på dusinvis av eksempler parallelt og har blitt aktivert på kreftcelle linjer og menneskelige svulster melanom, colon, prostata og glioblastom multiforme. Viser vi metodikken for seks kjerne histone modifikasjoner som representerer nøkkelkomponenter i epigenetic regulatoriske landskapet i menneskelig melanom linjer og svulst prøver. Disse endringene inkluderer H3K27ac (forsterker), H3K4me1 (aktiv og klar enhancers), H3K4me3 (arrangører), H3K79me2 (transkribert regioner), H3K27me3 (polycomb undertrykkelse), og H3K9me3 (heterochromatic undertrykkelse). Disse merkene kan brukes enten alene eller i kombinasjon for å identifisere funksjonelt forskjellige chromatin stater som representerer både undertrykkende og aktive domener.

<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

Histone endringene utgjør en viktig komponent i epigenome og spille viktige forskrifter rolle i å bestemme tilknyttet loci transcriptional status. I tillegg har tilstedeværelsen av endringer blitt brukt til å bestemme posisjonen og identitet ikke-koding funksjonelle elementer som enhancers. De siste årene, har chromatin immunoprecipitation etterfulgt av neste generasjons sekvensering (ChIP-seq) blitt et kraftig verktøy for å bestemme genomet hele profiler av personlige histone modifikasjoner. Det har imidlertid blitt stadig klarere at kombinasjon mønstre av chromatin modifikasjoner, referert til som Chromatin stater, fastslå identiteten og natur tilhørende genomisk locus. Derfor arbeidsflyter bestående av robust høy gjennomstrømming (HT) metoder for profilering flere histone endring merker, samt beregningsformelen analyser rørledninger i stand til å håndtere myriader av ChIP-Seq profilering datasett, er nødvendig for omfattende fastsettelse av epigenomic stater i mange eksempler. Den HT-ChIP-Seq arbeidsflyt presenteres her består av to moduler: 1) en eksperimentell protokoll for profilering flere histone endringer fra små mengder svulst prøver og cellelinjer i 96-brønnen format; og 2) en beregningsformelen data analyse rørledning som kombinerer eksisterende verktøy for å beregne både individuelle merke belegg og kombinasjon chromatin staten mønstre. Sammen lette disse to modulene enkel bearbeiding av hundrevis av ChIP-Seq eksempler på en rask og effektiv måte. Arbeidsflyten presenteres her brukes til å utlede chromatin staten mønstre fra 6 histone merke profiler i melanom svulster og linjer. Total, presenterer vi en omfattende ChIP-seq-arbeidsflyt som kan brukes til en rekke menneskelige svulst prøver og kreftcelle linjer å avgjøre epigenomic avvik i ulike malignancies.

Denne protokollen tillater immunoprecipitation fra frosne tumor vev og linjer som kan utføres på dusinvis av eksempler parallelt med en høy gjennomstrømming metode (figur 1A). Chromatin fragmenter burde omfang imellom ~ 200-1000 bps for optimal immunoprecipitation. Vi har bemerket at tiden det tar å oppnå samme klippe lengden varierer for ulike vev og celletyper. Chip fra små mengder vev avhenger holder lysate kulden alltid særlig und...

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 integrert plattform for genomet hele kartlegging av Chromatin stater med høy gjennomstrømming ChIP-sekvensering Tumor vev

Her beskriver vi en optimalisert høy gjennomstrømming ChIP-sekvensering protokollen og beregningsformelen analyser rørledning for fastsettelse av genomet hele chromatin staten mønstre fra frosne tumor vev og linjer.

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

The overall goal of this protocol is to unbiasedly identify combinatorial chromatin state patterns in tumor tissues and cancer cell lines. This method could help answer key questions in the cancer epigenetics field such as how a barren histone modification states could contribute to tumor genesis. The main advantage of this technique is that multiple histone modifications, as well as multiple samples could be processed both simultaneously, as well as efficiently.First, dissociate 50 milligrams of melanoma tissue manually and two milliliters of Hank's Balanced Salt Solution or HBSS, using a sterile razor blade. Mince the tissue into three or four millimeter pieces for approximately five minutes in a sterile tissue culture dish. Transfer the tissue solution to a dissociator tube and add another eighth milliliters of HBSS.Further dissociate the tissue, using a dissociator, until homogenized. Next, crosslink the tissue, adding 200 microliters of 16%formaldehyde per three milliliters of HBSS. Using a mixer, shake the mixture at 10 rpms for exactly 10 minutes at 37 degrees Celsius.After removing the sample from the incubator, add 200 microliters of tumoral glycine per three milliliters of sample and shake the mixture at 10 rpms for five minutes at 37 degrees Celsius. Spin the sample at 934 times g for five minutes at four degrees Celsius using a bench top centrifuge. When finished, remove the supernatant and add five milliliters of ice cold PBS.Then, centrifuge the sample again at 934 times g then remove the supernatant. Add 300 microliters of ChIP harvest buffer with protease inhibitors per 50 milligrams of tissue and lyse the samples for 30 minutes on ice. While the cells are lysing, turn on the water bath disruptor and associated cooling system and allow the temperature to reach four degrees Celsius.Place the sonicator tube in the water bath disruptor and sonicate the melanoma tissue for 60 cycles at 30 seconds on and 30 seconds off to obtain chromatin fragments of 200 to 600 base pairs. For eight milligrams of melanoma tissue, incubate three micrograms of each histone antibody with 20 microliters of protein g magnetic beads and 100 microliters of binding blocking buffer for two hours at four degrees Celsius with rotation, using a tube revolver. After sonication, dilute the sample five times using ChIP dilution buffer to bring the SDS concentration down to 0.1%After incubation of the antibody and protein g magnetic beads is complete, remove the ChIP dilution buffer and re suspend the beads in 240 microliters of the fresh buffer.Then, place 20 microliters aliquots of the beads into 12 separate tubes, each of which will be used for a separate tissue sample. Aliquot the sonicated material evenly to the protein g beads with specific antibody and rotate the tubes using a tube revolver overnight at four degrees Celsius. On the morning after reverse cross linking, transfer the antibody protein solution to a 96 well plate and place it on a magnetic stand.After allowing the beads to adhere for at least 30 seconds, remove the supernatant. Wash the beads five times with 150 microliters of ice cold RIPA wash buffer using a multi channel pipette. Move the magnet continuously for 30 seconds per wash, then remove the supernatant.After the washing steps, add an elution buffer master mix, containing 44 microliters of direct elution buffer, one microliter of Rnase and five microliters of protenase k per chip and input the sample for reverse cross linking. Incubate the samples using a PCR thermal cycler for four hours at 37 degrees Celsius, four hours at 50 degrees Celsius and eight to 16 hours at 65 degrees Celsius. On the next morning, place the samples back on the magnet and transfer the supernatant to a new 96 well PCR plate which contains the amino precipitated DNA.Add 2.3 x paramagnetic beads to the solution and carefully pipette up and down 25 times using a multi channel pipette. Following this, incubate at room temperature for four minutes. Then place the samples back on the magnet and incubate at room temperature for another four minutes.After discarding the supernatant, add 150 microliters of 70%ethanol to the samples and incubate at room temperature for 30 seconds without disturbing the beads. After allowing the beads to dry, remove the samples from the magnet and add 30 microliters of 10 millimolar traceal. Mix by pipetting 25 times.After incubating the samples at room temperature, place them back on the magnet and incubate at room temperature for another four minutes. Transfer 20 microliters of each sample to a new 96 well plate for library preparation. After PCR amplification, remove the samples and bring the total volume to 100 microliters using nucleus free water.Perform double sided paramagnetic size selection by first adding 55 microliters of beads to each sample and mixing 25 times with a pipette. Then incubate at room temperature for four minutes. Place the samples back on the magnet and incubate at room temperature for another four minutes to attain large fragments on the beads that are unsuitable for sequencing.After incubation, transfer the supernatant to a new 96 well PCR plate. Add another 25 microliters of paramagnetic beads and mix by pipetting 25 times and incubate at room temperature off the magnet for four minutes. After incubating at room temperature, place the samples back on the magnet and incubate at room temperature for four minutes.Then discard the supernatant. While leaving the samples on the magnet, add 150 microliters of 70%ethanol and incubate at room temperature for 30 seconds without disturbing the beads. Remove the ethanol and repeat the wash.After allowing the beads to dry, remove the samples from the magnet and add 25 microliters of 10 millimolar traceal pH 8.0. Mix by pipetting 25 times and incubate at room temperature, off the magnet for four minutes. After incubating at room temperature, place the samples back on the magnet and incubate at room temperature for four minutes.Now, quantify the libraries using a high sensitivity DNA electropherogram instrument before multiplexing to ensure the sizes are suitable for sequencing. This protocol allows immunoprecipitation of frozen tumor tissues and cell lines that can be performed on dozens of samples and parallel using a high throughput method. Chromatin fragments should range between 200 and 1000 base pairs for optimal immunoprecipitation.Purified ChIP DNA should be quantified before proceeding to library preparation. Completed libraries suitable for multiplexing and next generation sequencing should range between 200 and 600 base pairs and be devoid of any primer dimers. Upon post sequencing, there are many quality control metrics that should be met before proceeding to further steps of the pipeline, such as macs peak calling and chromHmm analysis.ChIP samples should be enriched over input DNA and each histone modification should display a distinct profile representing a specific component of the epigenetic landscape. The chrome HMM algorithm uses a Multivariate Hidden Markov Model to identify the most prominent reoccurring combinatorial and spacial chromatin patterns based on the histone modifications studied. Using these six modifications, chrome HMM identifies functionally distinct chromatin states representing both repressive and active domains, such as polycomb repression, heterochromatic repression, active transcription, and active enhancers.Once mastered, this technique can be done in three to four days, if it's performed properly. While attempting this procedure, it's important to determine optimal sonication conditions and specification for antibodies before continuing to library preparation and data analysis. For learning this procedure, other methods, such as RRS sequencing and chromosome confirmation capture can be performed to determine coalition of chromatin state parts with gene expression and higher chromatin structure.After the experiment, the technique has paved way for researchers in cancer epigenetics field to explore chromatin state patterns in patient tumor samples. After watching this video, you should have a good understanding of how to process and disassociate tumor tissues, perform chromatin immunoprecipitation, prepare libraries for nextgen sequecing, process post sequencing data and gain insights into initial steps of downstream analysis. Don't forget that when working with formaldehyde, it can be extremely hazardous and precautions, such as an appropriate fume hood, should always be taken when performing this procedure.

Research

JoVE Journal

Cancer Research

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De<em&gt; Drosophila</em&gt; Imaginal Disc Tumor Model: Visualisering og Kvantifisering av genekspresjon og Tumor Invasivitet Bruke Genetic Mosaikk

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Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

Etablering og karakterisering av små tarm Nevroendokrine tumor Spheroids

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer

Genomomfattende analyse av DNA-metylering i gastrointestinal kreft

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is ...

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

Pasientavledede tumor utviser som en "levende" preklinisk plattform for å forutsi legemiddelresistens hos pasienter

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...