We thank Chris Benner for implementing the X. tropicalis genome (xenTro2, xenTro2r) into HOMER and the Gilchrist lab for discussions on post-sequencing analysis. I.P. assisted the GO term analysis. G.E.G and J.C.S. were supported by the Wellcome Trust and are now supported by the Medical Research Council (program number U117597140).

<ol>
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The authors declare that they have no competing financial interests.

Unser Protokoll beschreibt, wie zu machen und zu analysieren, genomweite Chromatin Profile Xenopus-Embryonen. Es umfasst alle Schritte von vernetzenden Proteinen an endogenen Loci in vivo auf die Verarbeitung Millionen liest, die angereicherten genomische Websites in silico. Da immer mehr Genom Entwürfe zur Verfügung stehen, sollte dieses Protokoll auf andere Modell und nicht-Modellorganismen sein. Die wichtigsten experimentellen Teil, der dieses Protokoll von bisherigen Arbeit 8,31,33,34 stellt, ist die nach der Fixierung vor, um vernetzte Kerne zu extrahieren. Es ermöglicht die effiziente Chromatin Solubilisierung und Scher und einfach Upscaling. Zusammen mit verbesserten Wirkungsgraden von Bibliothek Vorbereitung dieses Protokoll erlaubt den Aufbau hochkomplexe ChIP-Seq-Bibliotheken von eineinhalb bis zwei Millionen Zellen der Chromatin-assoziierte Epitop von Interesse exprimieren. Für ChIP-qPCR-Experimente, einige zehntausend dieser Zellen sind in der Regel genug,für DNA Bereicherung auf vielleicht sechs verschiedenen genomischen Loci zu überprüfen. Diese Zahlen sind vorsichtige Schätzungen, kann aber je nach Expressionsniveau Protein, Antikörper Qualität abhängig, Vernetzungseffizienz und Epitop Zugänglichkeit. Als Richtwert ca. 4000 Zellen in der Mitte Blastulastadium (8,5 nach Nieuwkoop und Faber 29) enthält eine einzige Xenopus Embryo, 40.000 Zellen in der späten Gastrula-Stadium (12) und 100.000 Zellen in einem frühen Schwanzknospe Bühne (20). Die exakte Fixierung Zeit für eine effiziente Immunfällung muss empirisch durch ChIP-qPCR (§ 10) ermittelt werden. Im allgemeinen werden mehr Fixierungszeiten benötigt, wenn das Experiment beinhaltet X. laevis Embryonen frühen Entwicklungsstadien, und schwach (oder indirekte) DNA-Bindungseigenschaften. Es wird jedoch nicht empfohlen Befestigungs Xenopus Embryonen länger als 40 Minuten, oder Verarbeitung mehr Embryonen als angegeben (Abschnitt 3), Chromatin Scher wird weniger effizient. Es ist wichtig,jede Glycin nach der Fixierung als dieser gemeinsamen Schritt zum Abschrecken Formaldehyd Kernentnahme aus dotterreichen Embryonen sehr schwierig machen zu verwenden. Derzeit ist der Grund hierfür nicht bekannt. Es ist denkbar, dass die Formaldehyd-Glycin-Addukt ferner mit N-terminalen Amino-Gruppen oder Argininresten 35 reagiert. Der Antikörper ist der Schlüssel für ein Chip-Experiment und ausreichende Kontrollen müssen durchgeführt, um ihre Spezifität für das Epitop von Interesse zeigen werden (siehe Richtlinien Landt et al. 36). Wenn kein ChIP Grade Antikörper verfügbar ist, kann die Einführung von entsprechenden Epitop markierte Fusionsproteine ​​eine legitime Alternative sein, da diese Proteine ​​Bindungsstellen besetzen 37 endogen. In diesem Fall sind nicht injizierten Embryonen am besten, als negative Kontrolle eher als ein Chip mit unspezifischen Serum zu verwenden. Diese Strategie kann auch angewendet werden, wenn das Protein von Interesse auf einem niedrigen Niveau, was zu der schlechten Rückgewinnung des enri ausdrückenched DNA. Als zur Herstellung ChIP-Seq Bibliotheken, wegen der geringen Menge an DNA, im Gebrauch, ist es empfehlenswert, für Verfahren, die die Anzahl der Reinigungsschritte zu verringern und um eine Reaktion zu kombinieren, um den Verlust von DNA auf ein Minimum zu entscheiden. Die Adapter und Grundierungen müssen mit Multiplex-Sequenzierung und der NGS-Plattform kompatibel sind (siehe Tabelle der spezifischen Materialien / Geräte). Bei der Verwendung von Y-Adaptern (mit langen Einzelstrang-Arme), ist es wichtig vor, verstärken die Bibliothek drei bis fünf PCR-Runden vor size-Auswahl DNA-Insertionen (z. B. 100 bis 300 bp) durch Gelelektrophorese. Einzelsträngigen Enden verursachen DNA Fragmente heterogen migrieren. Testläufe mit verschiedenen Mengen an Ausgangs-DNA (zB 0,1, 0,5, 1, 2, 5, 10 und 20 ng) werden empfohlen, um die Gesamtzahl der PCR-Zyklen zu bestimmen (weniger als oder gleich 18 Zyklen) erforderlich, um eine Größe zu machen -Ausgewählte Bibliothek von 100 bis 200 ng. Verringerung der Anzahl der PCR-Zyklen macht die Sequenzierung redundant liest weniger wahrscheinlich. Die Festphasen-reversible Immobilisierung Perlen sind gute Reinigung Reagenzien zur effizienten Wiederherstellung der DNA von Interesse und zuverlässig entfernen Sie alle freien Adaptern und Dimere von Ligation und PCR-Reaktionen. In Bezug auf die Anzahl, Art und Länge der liest, um 20 bis 30 Millionen Single-End liest von 36 bp ist genug für die meisten ChIP-Seq-Experimente, die ganze Xenopus Genom mit ausreichender Tiefe zu decken. Die häufigsten NGS Maschinen sind routinemäßig in der Lage, die diese Kriterien erfüllen. Es kann jedoch von Vorteil sein, um die Anzahl zu erhöhen der liest, wenn eine breite Verteilung der liest wird erwartet, da mit Histon-Modifikationen zu beobachten, anstatt scharfe Spitzen. Für viele ChIP-Seq Experimenten 4 bis 5 unterschiedlich indizierten Bibliotheken gepoolt und in einer Durchflusszelle Fahrspur unter Verwendung einer Hochleistungsmaschine NGS sequenziert werden. Manchmal ist auch ratsam, die Lese Länge und Sequenz an beiden Enden der DNA-Vorlage (Paired-End), um Abbildbarkeit w erhöhen verlängernHenne Analyse Chromatin innerhalb repetitiven genomischen Regionen. Dieses Protokoll wurde erfolgreich in einer Vielzahl von Chromatin Funktionen wie Transkriptionsfaktoren, Signalmediatoren und posttranslationale Histonmodifizierungen aufgebracht. Allerdings Embryonen gewinnen einen zunehmenden Grad an zellulärer Heterogenität, wie sie sich entwickeln und Chromatin-Profile schwerer zu interpretieren. Erste Schritte auf in Arabidopsis und Drosophila gewebsspezifisch Profil Chromatin Landschaften durch Extrahieren zelltypspezifische Kerne 38,39 erzielt. Unser Protokoll umfasst eine Kernextraktionsschritt, der den Weg für die gewebespezifische ChIP-Seq anderer Embryonen bahnen könnte.

The first attempts to characterize protein-DNA interactions in vivo were reported about 30 years ago in an effort to understand RNA polymerase-mediated gene transcription in bacteria and in the fruit fly1,2. Since then, the use of immunoprecipitation to enrich distinct chromatin features (ChIP) has been widely adopted to capture binding events and chromatin states with high efficiency3. Subsequently, with the emergence of powerful microarray technologies, this method led to the characterization of genome-wide chromatin landscapes4. More recently, chromatin profiling has become even more comprehensive and high-resolution, because millions of co-immunoprecipitated DNA templates can now be sequenced in parallel and mapped to the genome (ChIP-Seq)5. As increasing numbers of genome assemblies are available, ChIP-Seq is an attractive approach to learn more about the genome regulation that underlies biological processes. 
Here we provide a protocol to perform ChIP-Seq on yolk-rich embryos such as those of the frog Xenopus. Drafts of the genomes of both widely used Xenopus species&#8212;X. tropicalis and X. laevis&#8212;have now been released by the International Xenopus Genome Consortium6. The embryos of Xenopus species share many desirable features that facilitate and allow the interpretation of genome-wide chromatin studies, including the production of large numbers of high-quality embryos, the large size of the embryos themselves, and their external development. In addition, the embryos are amenable to classic and novel manipulations like cell lineage tracing, whole-mount in situ hybridisation, RNA overexpression, and TALEN/CRISPR-mediated knockout technology.
The following protocol builds on the work of Lee et al., Blythe et al. and Gentsch et al.7-9. Briefly, Xenopus embryos are formaldehyde-fixed at the developmental stage of interest to covalently bind (cross-link) proteins to their associated genomic DNA. After nuclear extraction, cross-linked chromatin is fragmented to focus subsequent sequencing on specific genomic binding or modification sites, and to minimize the contributions of flanking DNA sequences. Subsequently, the chromatin fragments are immunoprecipitated with a ChIP-grade antibody to enrich those containing the protein of interest. The co-immunoprecipitated DNA is stripped from the protein and purified before creating an indexed (paired-end) library for next-generation sequencing (NGS). At the end, simple command lines are offered for the post-sequencing analysis of ChIP-Seq data.

<table><tbody><tr><td>1/16 inch tapered microtip</td><td>Qsonica</td><td>4417</td><td>This microtip is compatible with Sonicator 3000 from Misonix and Q500/700 from Qsonica.</td></tr><tr><td>8 ml glass sample vial with cap</td><td>Wheaton</td><td>224884</td><td>8 ml clear glass sample vials for aqueous samples with 15-425 size phenolic rubber-lined screw caps.</td></tr><tr><td>Adaptor</td><td>e.g., IDT or Sigma</td><td>NA</td><td>TruSeq universal adaptor, 
AATGATACGGCGACCACCGAG&lt;br /&gt;
ATCTACACTCTTTCCCTACAC&lt;br /&gt;
GACGCTCTTCCGATC*T. TruSeq indexed adaptor, 
P-GATCGGAAGAGCACACGTC&lt;br /&gt;
TGAACTCCAGTCAC &amp;#8208;NNNNNN&amp;#8208;&lt;br /&gt;
ATCTCGTATGCCGTCT&lt;br /&gt;TCTGCTT*G. *, phosphorothioate bondphosphate group at 5&amp;#39; end. NNNNNN, index (see TruSeq ChIP Sample Preparation Guide for DNA sequence). Order adaptors HPLC purified. Adaptors can be prepared by combining equimolar amounts (each 100 &amp;#181;M) of the universal and the indexed adaptor and cooling them down slowly from 95 &amp;#176;C to room temperature. Use 1.5 pmol per ng of input DNA. Store at -20 &amp;#176;C.</td></tr><tr><td>b2g4pipe (software)</td><td>Blast2GO</td><td>non-commercial</td><td>http://www.blast2go.com/data/blast2go/b2g4pipe_v2.5.zip</td></tr><tr><td>BLAST+ (software)</td><td>Camacho et al.</td><td>non-commercial</td><td>http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastDocs&amp;amp; &lt;br /&gt;DOC_TYPE=Download</td></tr><tr><td>Bowtie (software)</td><td>Langmead et al.</td><td>non-commercial</td><td>http://bowtie-bio.sourceforge.net/index.shtml</td></tr><tr><td>cisFinder (software)</td><td>Sharov et al.</td><td>non-commercial</td><td>http://lgsun.grc.nia.nih.gov/CisFinder/</td></tr><tr><td>Chip for capillary electrophoresis</td><td>Agilent Technologies</td><td>5067-1504</td><td>Load this chip with 1 &amp;micro;l DNA for library quality control. Store at 4 &amp;deg;C.</td></tr><tr><td>Chip-based capillary electrophoresis system</td><td>Agilent Technologies</td><td>G2940CA</td><td>The Agilent 2100 BioAnalyzer is used to check the quality of ChIP-Seq libraries. Keep reagents at 4 &amp;deg;C.</td></tr><tr><td>ChIP-Seq library preparation kit (KAPA Hyper Prep Kit)</td><td>Kapa Biosystems</td><td>KK8504</td><td>Kit contains KAPA end repair and A-tailing enzyme mix, end Repair and A-tailing buffer, DNA ligase, ligation buffer, KAPA HiFi HotStart ReadyMix (2X), and KAPA library amplification primer mix (10X) (see also PCR primers). Adaptors are not included. Store at -20 &amp;deg;C.</td></tr><tr><td>ChIP-Seq library preparation kit (alternative, ThruPLEX-FD Prep Kit)</td><td>Rubicon Genomics</td><td>R40048</td><td>Kit uses their own stem-loop adaptors and primers. This kit eliminates intermediate purification steps and is as sensitive as the KAPA Hyper Prep Kit. Store at -20 &amp;deg;C.</td></tr><tr><td>Cluster3 (software)</td><td>de Hoon et al.</td><td>non-commercial</td><td>http://bonsai.hgc.jp/~mdehoon/software/cluster</td></tr><tr><td>FastQC (software)</td><td>Simon Andrews</td><td>non-commercial</td><td>http://www.bioinformatics.babraham.ac.uk/projects/fastqc</td></tr><tr><td>Fluorometer</td><td>life technologies</td><td>Q32866</td><td>Qubit 2.0 Fluorometer</td></tr><tr><td>Fluorometer reagents</td><td>life technologies</td><td>Q32851</td><td>The kit provides concentrated assay reagent, dilution buffer, and pre-diluted DNA standards for the Qubit fluorometer. Store DNA standards at 4 &amp;deg;C, buffer and dye at room temperature.</td></tr><tr><td>Formaldehyde</td><td>Sigma</td><td>F8775-4X25ML</td><td>Formaldehyde solution, for molecular biology, 36.5-38% in H2O, stabilised with 10-15% methanol. Store at room temperature. CAUTION: Formaldehyde is corrosive and highly toxic.</td></tr><tr><td>Gel (E-Gel EX agarose , 2%)</td><td>life technologies</td><td>G4010</td><td>Pre-cast gel with 11 wells, openable format. Leave one lane between ladder and library empty to avoid cross-contamination. Store gels at room temperature.</td></tr><tr><td>Gel electrophoresis system</td><td>life technologies</td><td>G6465</td><td>E-Gel iBase and E-Gel Safe Imager combo kit for size-selecting ChIP-Seq libraries.</td></tr><tr><td>Gel extraction kit</td><td>Qiagen</td><td>28706</td><td>Store all reagents at room temperature. Use 500 &amp;micro;l of QG buffer per 100 mg of 2% agarose gel slice to extract DNA. Use MinElute columns (from MinElute PCR purification kit) to elute DNA twice.</td></tr><tr><td>HOMER (software)</td><td>Chris Benner</td><td>non-commercial</td><td>http://homer.salk.edu/homer/index.html</td></tr><tr><td>Hybridization oven</td><td>Techne</td><td>FHB1D</td><td>Hybridizer HB-1D</td></tr><tr><td>IGV (software)</td><td>Robinson et al.</td><td>non-commercial</td><td>http://www.broadinstitute.org/igv/home</td></tr><tr><td>Illumina CASAVA-1.8 quality filter (software)</td><td>Assaf Gordon</td><td>non-commercial</td><td>http://cancan.cshl.edu/labmembers/gordon/fastq_illumina_filter</td></tr><tr><td>Java TreeView (software)</td><td>Alok Saldanha</td><td>non-commercial</td><td>http://jtreeview.sourceforge.net</td></tr><tr><td>Laboratory jack</td><td>Edu-Lab</td><td>CH0642</td><td>This jack is used to elevate sample in sound enclosure for sonication.</td></tr><tr><td>Ladder, 100 bp</td><td>New England BioLabs</td><td>N3231</td><td>Keep 1x solution at room temperature. Store stock at -20 &amp;deg;C.</td></tr><tr><td>Ladder, 1 kb</td><td>New England BioLabs</td><td>N3232</td><td>Keep 1x solution at room temperature. Store stock at -20 &amp;deg;C.</td></tr><tr><td>Low-retention 1.5-ml microcentrifuge tubes</td><td>life technologies</td><td>AM12450</td><td>nonstick, RNase-free microfuge tubes, 1.5 ml</td></tr><tr><td>MACS2 (software)</td><td>Tao Liu</td><td>non-commercial</td><td>https://github.com/taoliu/MACS</td></tr><tr><td>Magnetic beads</td><td>life technologies</td><td>11201D</td><td>These Dynabeads are superparamagnetic beads with affinity purified polyclonal sheep anti-mouse IgG covalently bound to the bead surface. Store at 4 &amp;deg;C.</td></tr><tr><td>Magnetic beads</td><td>life technologies</td><td>11203D</td><td>These Dynabeads are superparamagnetic beads with affinity purified polyclonal sheep anti-rabbit IgG covalently bound to the bead surface. Store at 4 &amp;deg;C.</td></tr><tr><td>Magnetic beads</td><td>life technologies</td><td>10001D</td><td>These Dynabeads are superparamagnetic beads with recombinant protein A covalently bound to the bead surface. Store at 4 &amp;deg;C.</td></tr><tr><td>Magnetic beads</td><td>life technologies</td><td>10003D</td><td>These Dynabeads are superparamagnetic beads with recombinant protein G covalently bound to the bead surface. Store at 4 &amp;deg;C.</td></tr><tr><td>Magnetic rack</td><td>life technologies</td><td>12321D</td><td>DynaMag-2 magnet</td></tr><tr><td>MEME</td><td>Bailey et al.</td><td>non-commercial</td><td>http://meme.nbcr.net/meme/</td></tr><tr><td>Na3VO4</td><td>New England BioLabs</td><td>P0758</td><td>Sodium orthovanadate (100 mM) is a commonly used general inhibitor for protein phosphotyrosyl phosphatases. Store at -20 &amp;deg;C.</td></tr><tr><td>NaF</td><td>New England BioLabs</td><td>P0759</td><td>Sodium fluoride (500 mM) is commonly used as general inhibitor of phosphoseryl and phosphothreonyl phosphatases. Store at -20 &amp;deg;C.</td></tr><tr><td>NGS machine</td><td>Illumina</td><td>SY-301-1301</td><td>Genome Analyzer IIx</td></tr><tr><td>NGS machine (high performance)</td><td>Illumina</td><td>SY-401-2501</td><td>HiSeq</td></tr><tr><td>Normal serum (antibody control)</td><td>Santa Cruz Biotechnology</td><td>sc-2028</td><td>Use as control for goat polyclonal IgG antibodies in ChIP-qPCR experiments. Store at 4 &amp;deg;C.</td></tr><tr><td>Normal serum (antibody control)</td><td>Santa Cruz Biotechnology</td><td>sc-2025</td><td>Use as control for mouse polyclonal IgG antibodies in ChIP-qPCR experiments. Store at 4 &amp;deg;C.</td></tr><tr><td>Normal serum (antibody control)</td><td>Santa Cruz Biotechnology</td><td>sc-2027</td><td>Use as control for rabbit polyclonal IgG antibodies in ChIP-qPCR experiments. Store at 4 &amp;deg;C.</td></tr><tr><td>Nucleic acid staining solution</td><td>iNtRON</td><td>21141</td><td>Use RedSafe nucleic acid staining solution at 1:50,000. Store at room temperature.</td></tr><tr><td>Orange G</td><td>Sigma</td><td>O3756-25G</td><td>1-Phenylazo-2-naphthol-6,8-disulfonic acid disodium salt. Store at 4 &amp;deg;C.</td></tr><tr><td>PCR primers</td><td>e.g., IDT or Sigma</td><td></td><td>Primers to enrich adaptor-ligated DNA fragments by PCR: AATGATACGGCGACCACCGA*G and CAAGCAGAAGACGGCATACGA*G, phosphorothioate bond. Primers designed by Ethan Ford. Combine primers at 5 &amp;micro;M each. Use 5 &amp;micro;l in a 50 &amp;micro;l PCR reaction. Store at -20 &amp;deg;C.</td></tr><tr><td>MinElute PCR purification kit&amp;nbsp;</td><td>Qiagen</td><td>28006</td><td>for purification of ChIP-qPCR and shearing test samples. Store MinElute spin columns at 4 &amp;deg;C, all other buffers and collection tubes at room temperature.</td></tr><tr><td>Phenol:chloroform:isoamyl alcohol (25:24:1, pH 7.9)</td><td>life technologies</td><td>AM9730</td><td>Phenol:Chloroform:IAA (25:24:1) is premixed and supplied at pH 6.6. Use provided Tris alkaline buffer to raise pH to 7.9. Store at 4 &amp;deg;C. CAUTION: phenol:chloroform:isoamyl alcohol is corrosive, highly toxic and combustible.</td></tr><tr><td>Primer3 (software)</td><td>Steve Rozen &amp;amp; Helen Skaletsky</td><td>non-commercial</td><td>http://biotools.umassmed.edu/bioapps/primer3_www.cgi</td></tr><tr><td>Protease inhibitor tablets</td><td>Roche</td><td>11836170001</td><td>cOmplete, Mini, EDTA-free. Use 1 tablet per 10 ml. Store at 4 &amp;deg;C.</td></tr><tr><td>Protease inhibitor tablets</td><td>Roche</td><td>11873580001</td><td>cOmplete, EDTA-free. Use 1 tablet per 50 ml. Store at 4 &amp;deg;C.</td></tr><tr><td>Proteinase K</td><td>life technologies</td><td>AM2548</td><td>proteinase K solution (20 &amp;micro;g/&amp;micro;l). Store at -20 &amp;deg;C.</td></tr><tr><td>RNase A</td><td>life technologies</td><td>12091-039</td><td>RNase A (20 &amp;micro;g/&amp;micro;l). Store at room temperature.</td></tr><tr><td>Rotator</td><td>Stuart</td><td>SB3</td><td>Rotator SB3</td></tr><tr><td>SAMtools (software)</td><td>Li et al.</td><td>non-commercial</td><td>http://samtools.sourceforge.neta</td></tr><tr><td>Solid phase reversible immobilisation beads</td><td>Beckman Coulter</td><td>A63882</td><td>The Agencourt AMPure XP beads are used to minimise adaptor dimer contamination in ChIP-Seq libraries. Store at 4 &amp;deg;C.</td></tr><tr><td>Sonicator 3000</td><td>Misonix/Qsonica</td><td></td><td>Newer models are now available. Q125, Q500 or Q700 are all suitable for shearing crosslinked chromatin.</td></tr><tr><td>Sound enclosure</td><td>Misonix/Qsonica</td><td></td><td>optional: follow the manufacturer&#039;s recommendation to obtain the correct sound enclosure.</td></tr><tr><td>Thermomixer</td><td>eppendorf</td><td>22670000</td><td>Thermomixer for 24 x 1.5 mL tubes. Precise temperature control from 4 &amp;deg;C above room temperature to 99 &amp;deg;C.</td></tr></tbody></table>

genome-wide snapshot of chromatin regulators and states in xenopus embryos by chip-seq

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and organ formation during development. Determining the locations and levels of such chromatin features in vivo will provide valuable information about the spatio-temporal regulation of genomic elements, and will support aspirations to mimic embryonic tissue development in vitro. The most commonly used method for genome-wide and high-resolution profiling is chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). This protocol outlines how yolk-rich embryos such as those of the frog Xenopus can be processed for ChIP-Seq experiments, and it offers simple command lines for post-sequencing analysis. Because of the high efficiency with which the protocol extracts nuclei from formaldehyde-fixed tissue, the method allows easy upscaling to obtain enough ChIP material for genome-wide profiling. Our protocol has been used successfully to map various DNA-binding proteins such as transcription factors, signaling mediators, components of the transcription machinery, chromatin modifiers and post-translational histone modifications, and for this to be done at various stages of embryogenesis. Lastly, this protocol should be widely applicable to other model and non-model organisms as more and more genome assemblies become available.

Gleichwertige Ergebnisse liefern wie die hier vorgestellt werden erwartet, wenn das Protokoll wird gut ausgeführt und der Antikörper verwendet wird von ChIP-Grade-Qualität (siehe Diskussion). Dieses Protokoll ermöglicht die Gewinnung von Zellkernen aus Formaldehyd fixierten Xenopus-Embryonen und die effiziente Scher Chromatin durch Beschallung (1A-C). Geschert Chromatin zeigt eine asymmetrische Verteilung von DNA-Fragmenten vor allem im Bereich von 100 bis 1.000 bp und Peaking zwischen 300 und 500 bp (Abbildung 1C). Eine minimale 50 pg von immunpräzipitierten DNA wird benötigt, um erfolgreich zu machen eine indizierte Paired-End ChIP-Seq-Bibliothek mit ähnlich großen DNA-Inserts (Abbildung 2A). Die Bibliothek sollte weitgehend frei von Dimeren Adapter, die auf dem Elektropherogramm bei etwa 120 bp gesehen werden kann. Nach Sequenzierung-durch-Synthese, vorverarbeitet Lesewerte werden in das Genom (Abbildung 2B, C) ​​abgebildet. In einem erfolgreichen Versuch mit <em&gt; X. tropicalis Embryonen liest von 40 bp in der Regel 50 bis 70% der Einendmaschine eindeutig zum Genom Zusammenbau v7.1 mit maximal zwei Fehlpaarungen zugeordnet werden. Während einliest auszurichten ziemlich gleichmßig über das Genom, die Ausrichtung des Chips liest ergibt strangspezifische Anreicherung, die die Chromatinstruktur Merkmal von Interesse flankieren. Dies ist, da alle Fragmente werden von dem 5&#39;-Ende (2C) 25 sequenziert. Die Ausweitung der Ausrichtung in Leserichtung zu einer durchschnittlichen Fragmentgröße ermöglicht exakte Profile für einzelne Chromatin Funktionen wie Transkriptionsfaktor-Bindungsereignisse. Diese DNA-Belegungen erscheinen als Peaks, wenn in IGV oder einem anderen kompatiblen Genom-Browser visualisiert. Peak Anrufern wie MACs verwendet, um den Ort dieser Peaks (3A) zu bestimmen. Auf diese Weise Zehntausende von Bindungsstellen wurden in der X. bestimmt tropicalis-Genoms für die T-Box-Transkriptionsfaktoren wie VEGT 26. ChIP-qPCR Erfahgen sollte die lokale Anreicherung von ChIP-Seq (3B) gefunden zu bestätigen. ChIP-Seq-Experimente erlauben die Erkundung genomweiten Eigenschaften von Chromatin-Funktionen. Beispielsweise kann die Berechnung der Lese- Verteilung über genomischen Elemente wie Transkriptionsstart und Terminationsstellen keine räumliche Bindungspräferenzen rund Gene (3C) zu markieren. Ebenso ist eine Heatmap der Lese Distributionen zu Stoßstellen wird verwendet, um verschiedene Chromatin Features auf einen genomweiten Skala (3D) zu vergleichen. Bestimmte Transkriptionsfaktoren binden DNA sequenzspezifisch. De novo-Motiv-Analyse der genomischen DNA zugrunde liegenden Peaks können diese Art von Informationen, einschließlich Co-angereicherten Motive potentieller Kofaktoren (3E) abzurufen. Die große Mehrheit der Zielgene zeigen DNA-Belegung zu einem niedrigeren und nicht höhere Ebene (3F). Diese Skala freie Funktion scheint recht häufig bei transcription Faktoren und schlägt vor, daß nur ein kleiner Bruchteil der Zielgene sind direkt mit biologischer Relevanz 27,28 geregelt. Die Analyse der angereicherten GO Begriffe oder andere Eigenschaften wie beispielsweise die differentielle Expression von Zielgenen können ferner offenbaren Einblicke in die biologische Funktion des Chromatins Funktion im Xenopus Embryo (Figur 3G). <img alt="Figur 1" src="/files/ftp_upload/52535/52535fig1highres.jpg" /> Abbildung 1. Chromatin Immunopräzipitation Verfahren für Xenopus-Embryonen. (A) Die Embryonen sind Formaldehyd fixierten in der Entwicklungsphase von Interesse kovalent binden (vernetzen) alle Proteine ​​mit genomischer DNA assoziiert. Bei der Kerngewinnung (B), wird vernetzt Chromatin fragmentiert, um gezieltere genomischen DNA-Bindung oder Chromatinmodifizierung Websites durch Minimierung der flankierenden DNEine Sequenz (C). Anschließend werden die Chromatin-Fragmente mit einem Chip-Grade-Antikörper immunpräzipitiert zu denen, die das Epitop von Interesse (D) anzureichern. Die co-immunpräzipitiert DNA wird das Protein ausgezogen und vor dem Erstellen der ChIP Fragmentbibliothek für NGS (Abbildung 2) gereinigt (E). <a href="https://www.jove.com/files/ftp_upload/52535/52535fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieses Bild anzuzeigen.</a> <img alt="Abbildung 2" src="/files/ftp_upload/52535/52535fig2highres.jpg" /> Abbildung 2. ChIP-Seq Bibliothek Vorbereitung, Sequenzierung-durch-Synthese, die Kartierung und Spitzen Berufung. (A) Das Elektropherogramm zeigt eine gute ChIP-Seq-Bibliothek mit DNA-Matrizen von 250 bis 450 bp. Diese Vorlagen beinhalten die DNA-Insert von Interesse durch die universelle (58 bp) flankiert und die indexierte (63 bp) Adapter. (B) </strong&gt; Millionen von Clustern, wobei jeder Cluster mit identischen Vorlagen sind sequenziert Base für Base in Gegenwart von allen vier Nukleotiden besitzen, reversible, deutliche Fluorophor und identische Kündigungs Eigenschaften. Fluoreszenzbilder werden in Echtzeit verarbeitet werden, um entsprechenden Basen, die letztlich zusammengebaut in liest nennen. (C) nur liest, dass Karte eindeutig dem Xenopus Genom aufbewahrt. Da alle Fragmente werden von dem 5&#39;-Ende sequenziert, zur Kartierung der ChIP liest ergibt strangspezifischen Peaks, die die Chromatinstruktur Merkmal von Interesse flankieren. Dabei Spitzen Anrufer erkennen die Bereicherung durch die Immunpräzipitation stammt und erweitern das heißt zu einer durchschnittlichen Fragmentlänge genau zu lokalisieren Chromatin Funktionen. <a href="https://www.jove.com/files/ftp_upload/52535/52535fig2large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieses Bild anzuzeigen.</a> <img alt = &quot;3&quot; src = &quot;/ files / ftp_upload / 52.535 / 52535fig3highres.jpg&quot; /&gt; Figur 3 Ein Beispiel einer Post Sequenzierung Analyse und Visualisierung von Daten mittels des zygotischen T-Box-Transkriptionsfaktor VEGT (zVegT). Alle Lesezählungen hier dargestellt sind normalisiert auf 10 Millionen eindeutig abgebildet und nichtredundanten liest. (A) Auszug der genomweiten Profil zVegT verbindlich in X. tropicalis Gastrula Embryos (Stadium 11 bis 12,5 nach Nieuwkoop und Faber 29). Jeder Peak, eine Anhäufung von erweiterten liest, stellt eine Bindungsstelle. Diese Spitzen sind durch MACS2 mit einem falschen Discovery Rate (FDR) von weniger als 1% genannt. Jeder MESP-Gens zeigt sehr proximalen und stromauf zVegT Bindung, sondern nur mespa und mespb durch diese Stufe (RNA-Seq-Daten 30) ausgedrückt. (B) DNA Belegungsniveaus zVegT durch ChIP-qPCR an verschiedenen Loci bestimmt (einschließlich eines nicht -gebundenen Region 0,5 kb stromaufwärts von β-Aktin) Konfirm die spezifische Anreicherung von ChIP-Seq gefunden. Ergebnisse für mespa mit Peak genannt (roter Balken) in (A) vergleichen. Die DNA Belegungspegel als Prozentsatz der Eingang für beide, den Chip mit der VEGT Antikörper (polyklonaler Kaninchen-IgG-Isotyp) und dem Chip mit dem Antikörper-Kontrolle (normales Kaninchen IgG) visualisiert. Fehlerbalken geben die Standardabweichung von zwei biologischen Replikaten. (C) METAGENE Analyse zeigt Präferenz zVegT Bindung (Tags über 25 bp klassierten) mit dem Promotor in Bezug auf jede andere Genombereich um und in Gen Körpern. (D) Bewegungsradius zeigt k-Mittelwert Cluster (k = 5) DNA Auslastung der zVegT und Smad2 / Smad3 (ChIP-Seq Daten 31) bezogen auf alle zVegT gebundenen Regionen Gastrulastadium (Tags über 25 bp klassierte). Die Heatmap ist log 2 basiert und bei 5 Tags pro bp zentriert. (E) De-novo-Motiv-Analyse entdeckt die kanonische T-Box-Transkriptionsfaktor-Bindungsmotiv in 38% der zVegT-Regionen gebunden, wenn die zugrunde liegende Motiv Wertung ist auf einer Entdeckungsrate von 5% im Hintergrund Sequenzen. Die Dichte Karte zeigt höchsten Anreicherung für den T-Box-Motiv in der Mitte zVegT Bindungsstellen, wobei die kanonische Smad2 / Smad3 Bindungsmotiv kaum angereichert. (F) Histogramm zeigt DNA Belegungsniveaus zVegT, die für jedes Ziel-Gen berechnet werden von allen Gipfeln (+/- 200 bp) zwischen 5 kb stromaufwärts [-]. und 1 kb stromabwärts [+] der entsprechenden Transkriptionsstartstellen (G) Top 300 Gene, die mit höchsten DNA-Auslastung innerhalb -5 kb und 1 kb sind für biologische Prozesse der frühen Embryonalentwicklung bereichert. Diese GO Begriffe sind im Einklang mit der vermeintlichen Funktion zVegT. Der FDR auf einem zweiseitigen Fisher-Test basiert und für multiples Testen korrigiert. <a href="https://www.jove.com/files/ftp_upload/52535/52535fig3large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieses Bild anzuzeigen.</a>

Watch this Scientific Journal Video about Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq at JoVE.com

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

Die Frage, wie Chromatin Regulierungsbehörden und Chromatin-Staaten betreffen das Genom in vivo ist der Schlüssel zum Verständnis, wie frühe Zellschicksal Entscheidungen werden in den sich entwickelnden Embryo gemacht. ChIP-Seq-die beliebteste Ansatz zur Chromatin Funktionen auf globaler Ebene, wird hier für Xenopus Embryonen beschrieben untersuchen.

Genomweite Snapshot von Chromatin Regulierungsbehörden und Staaten in<em&gt; Xenopus</em&gt; Embryonen von ChIP-Seq

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and ...

genome-wide-snapshot-chromatin-regulators-states-xenopus-embryos-chip

Research

JoVE Journal

Developmental Biology

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

12.3K Aufrufe.  MRC National Institute for Medical Research.  Die Frage, wie Chromatin Regulierungsbehörden und Chromatin-Staaten betreffen das Genom in vivo ist der Schlüssel zum Verständnis, wie frühe Zellschicksal Entscheidungen werden in den sich entwickelnden Embryo gemacht. ChIP-Seq-die beliebteste Ansatz zur Chromatin Funktionen auf globaler Ebene, wird hier für Xenopus Embryonen beschrieben untersuchen. 

Serie: Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

Chromatin Immunopräzipitation (ChIP) Protokoll für Low-Fülle embryonalen Proben

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

Forschung

Genetik

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

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of associated loci. In addition, the presence of specific modifications has been used to determine the position and identity non-coding functional elements such as enhancers. In recent years, chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) has become a powerful tool in determining the genome-wide profiles of individual histone modifications. However, it has become increasingly clear that the combinatorial patterns of chromatin modifications, referred to as Chromatin States, determine the identity and nature of the associated genomic locus. Therefore, workflows consisting of robust high-throughput (HT) methodologies for profiling a number of histone modification marks, as well as computational analyses pipelines capable of handling myriads of ChIP-Seq profiling datasets, are needed for comprehensive determination of epigenomic states in large number of samples. The HT-ChIP-Seq workflow presented here consists of two modules: 1) an experimental protocol for profiling several histone modifications from small amounts of tumor samples and cell lines in a 96-well format; and 2) a computational data analysis pipeline that combines existing tools to compute both individual mark occupancy and combinatorial chromatin state patterns. Together, these two modules facilitate easy processing of hundreds of ChIP-Seq samples in a fast and efficient manner. The workflow presented here is used to derive chromatin state patterns from 6 histone mark profiles in melanoma tumors and cell lines. Overall, we present a comprehensive ChIP-seq workflow that can be applied to dozens of human tumor samples and cancer cell lines to determine epigenomic aberrations in various malignancies.

Here, we describe an optimized high-throughput ChIP-sequencing protocol and computational analyses pipeline for the determination of genome-wide chromatin state patterns from frozen tumor tissues and cell lines.

Eine integrierte Plattform für genomweite Kartierung von Chromatin Staaten mit hohem Durchsatz ChIP-Sequenzierung im Tumorgewebe

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

Krebsforschung

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.

Generation der genomweiten Chromatin Konformation Capture Bibliotheken aus dicht inszenierten Anfang Drosophila Embryos

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

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Epigenetische Regulation der Herz Differenzierung von embryonalen Stammzellen und Gewebe

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Entwicklungsbiologie

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Chromatin Immunopräzipitation von humanen embryonalen Stammzellen

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Biologie

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) is widely used to identify genome-wide binding locations of DNA-binding proteins. However, the ChIP-seq method is limited by its heterogeneity in length of sonicated DNA fragments and non-specific background DNA, resulting in low mapping resolution and uncertainty in DNA-binding sites. To overcome these limitations, the combination of ChIP with exonuclease digestion (ChIP-exo) utilizes 5&#8217; to 3&#8217; exonuclease digestion to trim the heterogeneously sized immunoprecipitated DNA to the protein-DNA crosslinking site. Exonuclease treatment also eliminates non-specific background DNA. The library-prepared and exonuclease-digested DNA can be sent for high-throughput sequencing. The ChIP-exo method allows for near base-pair mapping resolution with greater detection sensitivity and reduced background signal. An optimized ChIP-exo protocol for mammalian cells and next-generation sequencing is described below.

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

Precise determination of protein-binding locations across the genome is important for understanding gene regulation. Here we describe a&#160;genomic mapping method that treats chromatin-immunoprecipitated DNA with exonuclease digestion (ChIP-exo) followed by high-throughput sequencing. This method detects protein-DNA interactions with near base-pair mapping resolution and high signal-to-noise ratio in mammalian neurons.

Hochauflösende Kartierung von Protein-DNA-Wechselwirkungen in Mausstammzell-abgeleiteten Neuronen mit Chromatin-Immunpräzipitation-Exonuklease (ChIP-Exo)

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled ...

CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, epigenetic regulators, and post-translation modifications of histones that combinatorially influence the gene expression state of nearby genes. RNA has also been shown to interact with various proteins to regulate chromatin dynamics&#160;and gene expression. Chromatin-associated RNA immunoprecipitation (CARIP) followed by next-generation sequencing (CARIP-Seq) is a novel method to survey RNAs associated with chromatin proteins, while chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq) is a powerful genomics technique to map the location of post-translational modification of histones, transcription factors, and epigenetic modifiers on a global-scale in ES cells. Here, we describe methods to perform CARIP-Seq and ChIP-Seq, including library construction for next-generation sequencing, to generate global chromatin-associated RNA and epigenomic maps in ES cells.

Here, we describe methods to perform ChIP-Seq and CARIP-Seq, including library preparation for next-generation sequencing, to generate global epigenomic and chromatin-associated RNA maps in ES cells.

CARIP-Seq und ChIP-Seq: Methoden zur Ermittlung von Chromatin-assoziierter RNA und Protein-DNA-Wechselwirkungen in embryonale Stammzellen

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Chromatin Immunopräzipitation (ChIP) mit Drosophila Gewebe

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Generierung von High Quality Chromatin Immunpräzipitation DNA Vorlage für Hochdurchsatz-Sequenzierung (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Single-Cell Factor Localization on Chromatin using Ultra-Low Input Cleavage Under Targets and Release using Nuclease

Determining the binding locations of a protein on chromatin is essential for understanding its function and potential regulatory targets. Chromatin Immunoprecipitation (ChIP) has been the gold standard for determining protein localization for over 30 years and is defined by the use of an antibody to pull out the protein of interest from sonicated or enzymatically digested chromatin. More recently, antibody tethering techniques have become popular for assessing protein localization on chromatin due to their increased sensitivity. Cleavage Under Targets &amp; Release Under Nuclease (CUT&amp;RUN) is the genome-wide derivative of Chromatin Immunocleavage (ChIC) and utilizes recombinant Protein A tethered to micrococcal nuclease (pA-MNase) to identify the IgG constant region of the antibody targeting a protein of interest, therefore enabling site-specific cleavage of the DNA flanking the protein of interest. CUT&amp;RUN can be used to profile histone modifications, transcription factors, and other chromatin-binding proteins such as nucleosome remodeling factors. Importantly, CUT&amp;RUN can be used to assess the localization of either euchromatic- or heterochromatic-associated proteins and histone modifications. For these reasons, CUT&amp;RUN is a powerful method for determining the binding profiles of a wide range of proteins. Recently, CUT&amp;RUN has been optimized for transcription factor profiling in low populations of cells and single cells and the optimized protocol has been termed ultra-low input CUT&amp;RUN (uliCUT&amp;RUN). Here, a detailed protocol is presented for single-cell factor profiling using uliCUT&amp;RUN in a manual 96-well format.

CUT&#38;RUN and its variants can be used to determine protein occupancy on chromatin. This protocol describes how to determine protein localization on chromatin using single-cell uliCUT&#38;RUN.

Einzelzellfaktorlokalisierung auf Chromatin unter Verwendung von Ultra-Low Input Cleavage Under Targets und Freisetzung mit Nuclease

Determining the binding locations of a protein on chromatin is essential for understanding its function and potential regulatory targets. Chromatin ...

Genomweite Snapshot von Chromatin Regulierungsbehörden und Staaten in

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