Name: genome-wide snapshot of chromatin regulators and states in xenopus embryos by chip-seq
Uploaded: 2015-02-26T13:00:00
Duration: 10 min 23 s
Description: Watch this Scientific Journal Video about Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq at JoVE.com

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).

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The authors declare that they have no competing financial interests.

Vores protokol skitserer, hvordan man laver og analysere genom-dækkende kromatin profiler fra Xenopus embryoner. Den dækker alle trin fra tværbindende proteiner til endogene loci in vivo til behandling af millioner af læser repræsenterer berigede genomiske steder i silico. Da et stigende antal genom udkast er tilgængelige, bør denne protokol anvendelse på andre modeller og ikke-modelorganismer. Det vigtigste eksperimentelle afsnit, der sætter denne protokol, bortset fra tidligere arbejde 8,31,33,34, er den post-fiksering procedure at udvinde tværbundne kerner. Det letter effektiv kromatin solubilisering og udskæring og let opskalering. Sammen med forbedrede virkningsgrader på biblioteket forberedelse denne protokol giver mulighed for opførelse af høj kompleksitet chip-Seq biblioteker fra en halv til to millioner celler, der udtrykker kromatin-epitop af interesse. For chip qPCR forsøg, et par ti tusinde af disse celler er normalt nokat kontrollere, om DNA berigelse på måske seks distinkte genomiske loci. Disse tal er forsigtige skøn, men kan variere afhængigt af proteinekspression niveau antistof kvalitet, tværbindende effektivitet og epitop tilgængelighed. Som en guide, en enkelt Xenopus embryo indeholder omkring 4.000 celler i midten blastula etape (8.5 efter Nieuwkoop og Faber 29), 40.000 celler på det sene gastrula etape (12) og 100.000 celler på det tidlige tailbud etape (20). Den nøjagtige fiksering tid til effektiv immunpræcipitation skal bestemmes empirisk af Chip-qPCR (afsnit 10). I almindelighed er længere fikseringstidspunkter påkrævet, hvis forsøget involverer X. laevis embryoner, tidlige udviklingsstadier, og svage (eller indirekte) DNA-bindende egenskaber. Men er det ikke anbefales fastsættelse Xenopus embryoner længere end 40 min, eller behandling af flere fostre end angivet (afsnit 3), kromatin klipning bliver mindre effektiv. Det er vigtigt ikkeat bruge nogen glycin efter fiksering, da dette fælles skridt for quenching formaldehyd kan gøre nukleare ekstraktion fra æggeblomme-rige embryoner meget vanskelige. I øjeblikket er årsagen til dette ikke er kendt. Det er tænkeligt, at formaldehyd-glycin addukt yderligere reagerer med N-terminal amino-grupper eller argininrester 35. Antistoffet er nøglen til enhver chip eksperiment og tilstrækkelig kontrol skal udføres for at vise sin specificitet for epitop af interesse (se retningslinjer Landt et al. 36). Hvis der ikke chip-grade antistof er tilgængelig, kan indførelsen af tilsvarende epitop-mærkede fusionsproteiner være en legitim alternativ, da disse proteiner kan indtage endogen bindingssteder 37. I dette tilfælde injicerede embryoner er bedst at bruge som en negativ kontrol snarere end en chip med non-specifik serum. Denne strategi kan også anvendes, hvis proteinet af interesse udtrykkes ved lave niveauer, hvilket resulterer i dårlig genvinding af EnriChed DNA. Som for at gøre chip Seq biblioteker, på grund af den lille mængde DNA i brug, anbefales det at vælge procedurer, som reducerer antallet af rensningstrin og at kombinere reaktioner at holde tab af DNA på et minimum. De adaptere og primere skal være forenelige med multiplex-sekventering og NGS platform (se tabel for specifikke materialer / udstyr). Hvis anvendelse af Y-adaptere (indeholdende lange enkeltstrengede arme), er det afgørende at pre-amplificere biblioteket med 3-5 runder af PCR før størrelsesmæssige vælge DNA-inserter (f.eks. 100 til 300 bp) ved gelelektroforese. Enkeltstrengede ender forårsager DNA-fragmenter til at migrere heterogent. Trial kører med forskellige mængder af input DNA (fx 0,1, 0,5, 1, 2, 5, 10 og 20 ng) anbefales for at bestemme det samlede antal PCR-cyklusser (mindre end eller lig med 18 cyklusser), der kræves til at foretage en størrelse -selected bibliotek på 100 til 200 ng. Reduktion af antallet af PCR-cyklusser gør sekventering af Redundant læser mindre sandsynligt. Solid fase reversible immobiliseringstrin perler er gode oprydning reagenser til effektivt genvinde DNA&#39;et af interesse og pålideligt fjerne frie adaptere og dimerer fra ligering og PCR-reaktioner. Med hensyn til antal, type og længde læser, omkring 20 til 30.000.000 enkelt ende læser på 36 bp er nok for de fleste chip-Seq eksperimenter at dække hele Xenopus genomet med tilstrækkelig dybde. De mest anvendte NGS maskiner er rutinemæssigt stand til at opfylde disse kriterier. Dog kan det være fordelagtigt at øge antallet af læser hvis der forventes en bred fordeling af læser, som observeret med histon modifikationer, snarere end skarpe toppe. For mange chip Seq eksperimenter, kan 4-5 forskelligt indekserede biblioteker samles og sekventeret i en strømningscelle bane ved hjælp af en højtydende NGS maskine. Nogle gange er også tilrådeligt at udvide den læste længde og sekvens begge ender af DNA-skabelonen (parret ende) for at øge mappability whøne analysere chromatin inden gentagne genomiske regioner. Denne protokol er blevet anvendt med succes til en bred vifte af kromatin funktioner såsom transkriptionsfaktorer, signalering mediatorer og posttranslationelle histon modifikationer. Men embryoner erhverve en stigende grad af cellulær heterogenitet som de udvikler og kromatin profiler bliver sværere at fortolke. Lovende skridt er allerede taget i Arabidopsis og Drosophila til tissue-specifikt profil kromatin landskaber ved at udvinde celletype-specifikke kerner 38,39. Vores Protokollen indeholder et skridt nuklear ekstraktion, som kunne bane vejen for vævsspecifikke chip-Seq i andre embryoner.

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 border="0" cellpadding="0" cellspacing="0">
	<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>IDT or Sigma</td>
			<td>NA</td>
			<td>TruSeq universal adaptor, 
AATGATACGGCGACCACCGAG 
ATCTACACTCTTTCCCTACAC 
GACGCTCTTCCGATC*T. TruSeq indexed adaptor, 
P-GATCGGAAGAGCACACGTC 
TGAACTCCAGTCAC &#8208;NNNNNN&#8208; 
ATCTCGTATGCCGTCT TCTGCTT*G. *, phosphorothioate bondphosphate group at 5&#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 &#181;M) of the universal and the indexed adaptor and cooling them down slowly from 95 &#176;C to room temperature. Use 1.5 pmol per ng of input DNA. Store at -20 &#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&#38;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 &#181;l DNA for library quality control. Store at 4 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#181;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;C.</td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>e.g., IDT or Sigma</td>
			<td>NA</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 &#181;M each. Use 5 &#181;l in a 50 &#181;l PCR reaction. Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>MinElute PCR purification kit&#160;</td>
			<td>Qiagen</td>
			<td>28006</td>
			<td>for purification of ChIP-qPCR and shearing test samples. Store MinElute spin columns at 4 &#176;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 &#176;C. CAUTION: phenol:chloroform:isoamyl alcohol is corrosive, highly toxic and combustible.</td>
		</tr>
		<tr>
			<td>Primer3 (software)</td>
			<td>Steve Rozen &#38; 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 &#176;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 &#176;C.</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>life technologies</td>
			<td>AM2548</td>
			<td>proteinase K solution (20 &#181;g/&#181;l). Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>life technologies</td>
			<td>12091-039</td>
			<td>RNase A (20 &#181;g/&#181;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 &#176;C.</td>
		</tr>
		<tr>
			<td>Sonicator 3000</td>
			<td>Misonix/Qsonica</td>
			<td>NA</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>NA</td>
			<td>optional: follow the manufacturer&#39;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 &#176;C above room temperature to 99 &#176;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.

Tilsvarende resultater til dem, der præsenteres her, er forventes, hvis protokollen er velgennemført og antistoffet i anvendelse er chip-grade kvalitet (se diskussion). Denne protokol tillader udvinding af kerner fra formaldehyd-fikseret Xenopus embryoner og effektiv klipning af chromatin ved lydbehandling (figur 1A-C). Skåret kromatin viser en asymmetrisk fordeling af DNA-fragmenter hovedsagelig i området fra 100 til 1.000 bp og topper mellem 300 og 500 bp (figur 1C). En minimal 50 pg immunopræcipiteret DNA kræves for en vellykket foretage en indekseret parret ende chip Seq bibliotek med samme størrelse DNA-inserter (figur 2A). Biblioteket bør være stort set blottet for adaptordimerer, som kan ses på elektroferogram på ca. 120 bp. Ved sekventering-by-syntese, forbehandlet læser mappes på genomet (figur 2B, C). I et vellykket eksperiment med <em&gt; X. tropicalis embryoner normalt 50 til 70% af en enkelt ende læser 40 bp kan kortlægges entydigt til genomet samling af V7.1 med maximalt to fejlparringer. Mens input læser tilpasse helt ensartet i hele genomet, tilpasning af chip læser resultater i streng-specifikke berigelser, der flankerer kromatin funktionen af ​​interesse. Dette er fordi alle fragmenter sekventeret fra 5&#39;-enden (figur 2C) 25. Udvidelse af tilpasningen i at læse retning til en gennemsnitlig fragment størrelse producerer nøjagtige profiler for enlige kromatin funktioner såsom transkriptionsfaktorbindende begivenheder. Disse DNA occupancies vises som toppe når visualiseret i IGV eller enhver anden kompatibel genom browser. Peak opkaldere som MACS anvendes til at bestemme placeringen af disse toppe (figur 3A). På denne måde titusinder af bindingssteder er fastlagt i X. tropicalis genomet for T-box transkriptionsfaktorer, såsom Vegt 26. Chip-qPCR erfamenter skal bekræfte lokale berigelse fundet af chip Seq (figur 3B). CHIP-Seq eksperimenter tillader udforske genom-dækkende karakteristika kromatin funktioner. For eksempel kan beregningen af læse- fordeling over genomiske elementer som transkriptionsstart og termineringssteder fremhæve eventuelle rumlige bindende præferencer omkring gener (figur 3C). Tilsvarende er en Heatmap af læste distributioner på højeste steder bruges til at sammenligne forskellige kromatin funktioner til en genom-plan (figur 3D). Visse transkriptionsfaktorer binder DNA-sekvens-specifikt. De novo motiv analyse af genomisk DNA underliggende toppe kan hente den slags oplysninger, herunder co-berigede motiver af potentielle co-faktorer (Figur 3E). Det store flertal af målgener viser DNA belægning på et lavere snarere end højere niveau (figur 3F). Denne skala-fri funktion synes at være ganske udbredt blandt transcription faktorer og foreslår, at kun en lille brøkdel af target gener er direkte reguleret med biologisk relevans 27,28. Analysen af berigede GO vilkår eller andre egenskaber, såsom den differentielle ekspression af målgener kan endvidere give indsigt i den biologiske funktion af kromatin funktion i Xenopus embryo (figur 3G). <img alt="Figur 1" src="/files/ftp_upload/52535/52535fig1highres.jpg" /> Figur 1. Chromatin immunfældning procedure for Xenopus embryoer. (A) embryoner formaldehyd-fikseret på udviklingsstadiet af interesse for kovalent binder (cross-link) eventuelle proteiner associeret med genomisk DNA. Ved nuklear ekstraktion (B), er tværbundet kromatin fragmenteret at indsnævre genomisk DNA binding eller kromatin modifikationssteder ved at minimere flankerende DNEn sekvens (C). Efterfølgende kromatinet fragmenter immunfældet med en chip-grade antistof at berige dem, der indeholder epitopen af interesse (D). Den co-immunpræcipiteret DNA krængede proteinet og oprenses (E), inden du opretter chippen fragment bibliotek for NGS (figur 2). <a href="https://www.jove.com/files/ftp_upload/52535/52535fig1large.jpg" target="_blank">Klik her for at se en større udgave af dette tal.</a> <img alt="Figur 2" src="/files/ftp_upload/52535/52535fig2highres.jpg" /> Figur 2. chip Seq bibliotek forberedelse, sekventering-by-syntese, kortlægning og peak kald. (A) elektroferogram viser en god Chip-Seq bibliotek med DNA-skabeloner af på 250 til 450 bp. Disse skabeloner medfører DNA-insert af interesse flankeret af Universal (58 bp) og indekseret (63 bp) adapter. (B) </strong&gt; Millioner af klynger, hvor hver klynge indeholder identiske skabeloner, er sekventeret base ved base i nærvær af alle fire nukleotider besidder reversible, tydelig fluorofor og identiske opsigelse egenskaber. Fluorescerende billeder bearbejdes i realtid til at ringe tilsvarende baser, som i sidste ende er samlet i læser. (C) Kun læser dette kort unikt til Xenopus genomet holdes. Da alle fragmenter sekventeret fra 5&#39;-enden, kortlægning af chip læser resulterer i streng-specifikke toppe, der flankerer kromatin funktionen af ​​interesse. Derved peak opkaldere registrere berigelse, der stammer fra immunpræcipitering og udvide læser til en gennemsnitlig længde fragment til præcist lokalisere kromatin funktioner. <a href="https://www.jove.com/files/ftp_upload/52535/52535fig2large.jpg" target="_blank">Klik her for at se en større udgave af dette tal.</a> <img alt = &quot;Figur 3&quot; src = &quot;/ files / ftp_upload / 52535 / 52535fig3highres.jpg&quot; /&gt; Figur 3. Et eksempel på post-sekventeringsanalyse og data visualisering ved hjælp af zygotisk T-box transkriptionsfaktor Vegt (zVegT). Alle læste tæller vist her er normaliseret til 10 millioner entydigt kortlagt og ikke-redundant læser. (A) Uddrag af hele genomet profil zVegT bindende i X. tropicalis gastrula embryoner (stadie 11-12,5 efter Nieuwkoop og Faber 29). Hver top, en bunke-up af udvidet læser, udgør en bindingssted. Disse toppe bliver kaldt af MACS2 med en falsk opdagelse sats (FDR) på mindre end 1%. Hver MESP gen viser meget proksimale og opstrøms zVegT bindende, men kun mespa og mespb udtrykkes af denne fase (RNA-Seq data 30). (B) DNA belægningsniveauer af zVegT som bestemt af Chip-qPCR på flere loci (herunder en ikke -bundet region 0,5 kb opstrøms for β-actin) fortroligRM specifik berigelse fundet af chip Seq. Sammenlign resultater for mespa med peak kaldet (rød bjælke) i (A). DNA belægning niveau visualiseres som en procentdel af input til både chippen med Vegt antistof (polyklonalt kanin-IgG-isotype) og chippen med antistoffet kontrol (normal kanin IgG). Fejlbjælker afspejler standardafvigelsen af to biologiske replikater. (C) Metagene analyse viser præferentiel zVegT binding (tags sammenflettede over 25 bp) til promotoren i forhold til enhver anden genomisk region rundt om og inden gen organer. (D) Heatmap viser K-middelværdi klynger (k = 5) DNA belægningsniveauer (tags arkiveret lodret over 25 bp), i zVegT og Smad2 / Smad3 (chip-Seq data 31) i forhold til alle zVegT-bundne regioner på gastrula stadium. Den Heatmap logge 2 baseret og centreret ved 5 mærker pr bp. (E) De novo motiv analyse opdager den kanoniske T-box transskriptionsfaktorbindende motiv i 38% af zVegT-bundne regioner, hvis den underliggende motiv score er normaliseret til en opdagelse sats i baggrunden sekvenser 5%. Tætheden kort viser højeste berigelse for T-box-motiv i centrum af zVegT bindingssteder, mens den kanoniske Smad2 / Smad3 bindingsmotiv næppe er beriget. (F) Histogram viser DNA belægningsniveauer af zVegT, som er beregnet for hver målgen fra alle toppe (+/- 200 bp) mellem 5 kb opstrøms [-]. og 1 kb nedstrøms [+] af tilsvarende transcription start sites (G) Top 300 gener med højeste DNA belægningsniveauer inden -5 kb og +1 kb er beriget for biologiske processer i den tidlige fosterudvikling. Disse går vilkår er i overensstemmelse med den formodede funktion zVegT. Den FDR er baseret på en to-tailed Fishers eksakte test og korrigeret for multipel testning. <a href="https://www.jove.com/files/ftp_upload/52535/52535fig3large.jpg" target="_blank">Klik her for at se en større udgave af dette tal.</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

Spørgsmålet om, hvordan kromatin tilsynsmyndigheder og kromatin stater påvirker genomet in vivo er nøglen til vores forståelse af, hvordan tidlig celle skæbne beslutninger træffes i det udviklende foster. Chip Seq-den mest populære metode til at undersøge kromatin funktioner på globalt plan-er skitseret her Xenopus embryoner.

Genom-dækkende Snapshot af Chromatin Regulators og stater i<em&gt; Xenopus</em&gt; Embryoner af 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 ...

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Developmental Biology

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

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

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Manipulation og<em&gt; In vitro</em&gt; Modning af<em&gt; Xenopus laevis</em&gt; Oocytter, efterfulgt af ICSI, for at studere fosterudviklingen

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

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Nøjagtig og Phenol Gratis DNA Kønsselektion Dag 30 soembryoner ved PCR

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Technique to Target Microinjection to the Developing Xenopus Kidney

Technique to Target Microinjection to the Developing Xenopus Kidney

Teknik Target Mikroinjektion til udviklingslandene<em&gt; Xenopus</em&gt; Nyre

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

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Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

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The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

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Visualisering og kvantitativ analyse af embryonisk angiogenese i<em&gt; Xenopus tropicalis</em

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Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

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Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

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The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

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Mikroinjektion af DNA i øjen knopper i xenopus laevis embryoner og billeddannelse af Gfp udtrykker optiske Axonal Arbors i intakt, levende xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...