Spørsmålet om hvordan kromatin regulatorer og kromatin stater påvirker genomet in vivo er nøkkelen til vår forståelse av hvor tidlig celle skjebne beslutninger fattes i utvikling av embryo. ChIP-Seq-den mest populære tilnærming for å undersøke kromatin funksjoner på et globalt nivå-er skissert her for Xenopus embryoer.
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.
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—X. tropicalis and X. laevis—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.
Vår protokoll skisserer hvordan å lage og analysere genom kromatin profiler fra Xenopus embryoer. Den dekker alle trinn fra tverrbindende proteiner til endogene loci in vivo for å behandle millioner av leser representerer beriket genomiske steder i silico. Siden stadig flere genom utkast er tilgjengelig, bør denne protokollen være aktuelt å modell for andre og ikke-modellorganismer. Det viktigste eksperimentelle delen, som setter denne protokollen bortsett fra tidligere arbeid 8,31,33,34, er den post-fiksering prosedyre for å trekke krysskoblede kjerner. Det muliggjør effektiv kromatin løseliggjøring og klipping og enkel oppskalering. Sammen med forbedrede effektiviteten av bibliotek forberedelse denne protokollen tillater bygging av høy kompleksitet chip-Seq biblioteker fra en halv til to millioner celler som uttrykker kromatin-assosiert epitop av interesse. For ChIP-qPCR eksperimenter, noen ti tusen av disse cellene er normalt nokfor å se etter DNA berikelse på kanskje seks forskjellige genomisk loci. Disse tallene er konservative anslag, men kan variere avhengig av proteinekspresjon nivå, antistoff kvalitet, tverrbindingseffektivitet, og epitop tilgjengelighet. Som en veiledning, inneholder et enkelt Xenopus embryo omtrent 4000 celler ved midten blastula trinn (8,5 etter Nieuwkoop og Faber 29), 40 000 celler ved slutten gastrulastadiet (12) og 100 000 celler på et tidlig stadium tailbud (20).
Den nøyaktige fiksering tid for effektiv immunoprecipitation må bestemmes empirisk av Chip-qPCR (§ 10). Generelt er lengre festetider nødvendig hvis forsøket innebærer X. laevis embryoer, tidlige utviklingsstadier, og svake (eller indirekte) DNA-bindende egenskaper. Men det er ikke anbefalt å fikse Xenopus embryo lenger enn 40 min, eller behandler flere embryoer enn angitt (§ 3), som kromatin klipping blir mindre effektiv. Det er viktig ikkeå bruke noen glysin etter fiksering som denne felles skritt for å slukke formaldehyd kan gjøre kjernekraft utvinning fra plommerike embryo svært vanskelige. For tiden, er grunnen til dette ikke er kjent. Det er tenkelig at formaldehyd-glycin-addukt reagerer videre med N-terminal amino-grupper eller argininrester 35.
Antistoffet er nøkkelen til enhver ChIP eksperiment og tilstrekkelig kontroller må bli utført for å vise dens spesifisitet for epitopen av interesse (se veiledning av Landt et al. 36). Hvis ingen ChIP klasse antistoff er tilgjengelig, kan innføring av tilsvarende epitop-tagget fusjonsproteiner være en legitim alternativ som disse proteinene kan oppta endogene bindingsseter 37. I dette tilfellet, uinjiserte embryoer som er best å bruke som en negativ kontroll i stedet for en brikke med ikke-spesifikk serum. Denne strategi kan også anvendes dersom proteinet av interesse er uttrykt ved lave nivåer som resulterer i dårlig gjenvinning av EnriChed DNA.
Som for å lage chip-Seq biblioteker, på grunn av den lave mengden av DNA i bruk, anbefales det å velge prosedyrer som reduserer antall rengjøringstrinn og å kombinere reaksjoner for å holde tap av DNA på et minimum. Adaptere og primere må være kompatibel med multiplex sekvensering og NGS platform (se tabell av konkrete materialer / utstyr). Ved hjelp av Y-adaptere (inneholdende lange enkelt-trådede armer), er det avgjørende å pre-amplifisere biblioteket med 3-5 runder med PCR-størrelse før valg av DNA-innskudd (f.eks., 100 til 300 bp) ved gelelektroforese. Enkelttrådete ender forårsake DNA-fragmenter å migrere heterogent. Prøvekjøringer med forskjellige mengder av inngangs DNA (for eksempel, 0,1, 0,5, 1, 2, 5, 10 og 20 ng) anbefales for å bestemme det totale antall PCR-sykluser (mindre enn eller lik 18 sykluser) som kreves for å gjøre en størrelse -selected bibliotek på 100 til 200 ng. Redusere antall PCR-sykluser gjengir sekvensering av redundant leser mindre sannsynlig. Solid fase reversible immobilisering perler er god rydde opp reagenser for å effektivt gjenopprette DNA av interesse og pålitelig fjerne noen gratis adaptere og dimerer fra ligering og PCR reaksjoner.
I form av antall, type og lengde av lyder, rundt 20-30 millioner single-end leser av 36 bp er nok for de fleste chip-Seq eksperimenter for å dekke hele Xenopus genomet med tilstrekkelig dybde. De mest utbredte NGS maskiner er rutinemessig stand til å møte disse kriteriene. Imidlertid kan det være fordelaktig å øke antall lyder hvis en bred fordeling av lyder er forventet, som observert med histonmodifikasjonene, heller enn skarpe topper. For mange chip-Seq eksperimenter, kan 4-5 annerledes indekserte bibliotekene samles og sekvensert i en flyt celle kjørefelt ved hjelp av en høy-ytelse NGS maskin. Noen ganger er også tilrådelig å forlenge lese lengde og sekvens begge ender av DNA-templatet (sammenkoblet ende) for å øke mappability whøne analysere kromatin innenfor repetitive genomiske regioner.
Denne protokollen har blitt brukt med hell til et bredt spekter av kromatin funksjoner som transkripsjonsfaktorer, signal meklere og posttranslasjonelle histonmodifikasjonene. Men embryoer erverve en økende grad av cellulær heterogenitet som de utvikler og kromatin profiler blir vanskeligere å tolke. Lovende tiltak har blitt gjort i Arabidopsis og Drosophila til vev-spesifikt profil kromatin landskap ved å trekke ut celletypespesifikke kjerner 38,39. Vår Protokollen inkluderer en kjernefysisk utvinning skritt, noe som kan bane vei for vevsspesifikt ChIP-Seq i andre embryoer.
The authors have nothing to disclose.
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).
1/16 inch tapered microtip | Qsonica | 4417 | This microtip is compatible with Sonicator 3000 from Misonix and Q500/700 from Qsonica. |
8 ml glass sample vial with cap | Wheaton | 224884 | 8 ml clear glass sample vials for aqueous samples with 15-425 size phenolic rubber-lined screw caps. |
Adaptor | IDT or Sigma | NA | TruSeq universal adaptor,
AATGATACGGCGACCACCGAG ATCTACACTCTTTCCCTACAC GACGCTCTTCCGATC*T. TruSeq indexed adaptor, P-GATCGGAAGAGCACACGTC TGAACTCCAGTCAC ‐NNNNNN‐ ATCTCGTATGCCGTCT TCTGCTT*G. *, phosphorothioate bondphosphate group at 5' 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 µM) of the universal and the indexed adaptor and cooling them down slowly from 95 °C to room temperature. Use 1.5 pmol per ng of input DNA. Store at -20 °C. |
b2g4pipe (software) | Blast2GO | non-commercial | http://www.blast2go.com/data/blast2go/b2g4pipe_v2.5.zip |
BLAST+ (software) | Camacho et al. | non-commercial | http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastDocs&DOC_TYPE=Download |
Bowtie (software) | Langmead et al. | non-commercial | http://bowtie-bio.sourceforge.net/index.shtml |
cisFinder (software) | Sharov et al. | non-commercial | http://lgsun.grc.nia.nih.gov/CisFinder/ |
Chip for capillary electrophoresis | Agilent Technologies | 5067-1504 | Load this chip with 1 µl DNA for library quality control. Store at 4 °C. |
Chip-based capillary electrophoresis system | Agilent Technologies | G2940CA | The Agilent 2100 BioAnalyzer is used to check the quality of ChIP-Seq libraries. Keep reagents at 4 °C. |
ChIP-Seq library preparation kit (KAPA Hyper Prep Kit) | Kapa Biosystems | KK8504 | 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 °C. |
ChIP-Seq library preparation kit (alternative, ThruPLEX-FD Prep Kit) | Rubicon Genomics | R40048 | 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 °C. |
Cluster3 (software) | de Hoon et al. | non-commercial | http://bonsai.hgc.jp/~mdehoon/software/cluster |
FastQC (software) | Simon Andrews | non-commercial | http://www.bioinformatics.babraham.ac.uk/projects/fastqc |
Fluorometer | life technologies | Q32866 | Qubit 2.0 Fluorometer |
Fluorometer reagents | life technologies | Q32851 | The kit provides concentrated assay reagent, dilution buffer, and pre-diluted DNA standards for the Qubit fluorometer. Store DNA standards at 4 °C, buffer and dye at room temperature. |
Formaldehyde | Sigma | F8775-4X25ML | 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. |
Gel (E-Gel EX agarose , 2%) | life technologies | G4010 | 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. |
Gel electrophoresis system | life technologies | G6465 | E-Gel iBase and E-Gel Safe Imager combo kit for size-selecting ChIP-Seq libraries. |
Gel extraction kit | Qiagen | 28706 | Store all reagents at room temperature. Use 500 µl 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. |
HOMER (software) | Chris Benner | non-commercial | http://homer.salk.edu/homer/index.html |
Hybridization oven | Techne | FHB1D | Hybridizer HB-1D |
IGV (software) | Robinson et al. | non-commercial | http://www.broadinstitute.org/igv/home |
Illumina CASAVA-1.8 quality filter (software) | Assaf Gordon | non-commercial | http://cancan.cshl.edu/labmembers/gordon/fastq_illumina_filter |
Java TreeView (software) | Alok Saldanha | non-commercial | http://jtreeview.sourceforge.net |
Laboratory jack | Edu-Lab | CH0642 | This jack is used to elevate sample in sound enclosure for sonication. |
Ladder, 100 bp | New England BioLabs | N3231 | Keep 1x solution at room temperature. Store stock at -20 °C. |
Ladder, 1 kb | New England BioLabs | N3232 | Keep 1x solution at room temperature. Store stock at -20 °C. |
Low-retention 1.5-ml microcentrifuge tubes | life technologies | AM12450 | nonstick, RNase-free microfuge tubes, 1.5 ml |
MACS2 (software) | Tao Liu | non-commercial | https://github.com/taoliu/MACS |
Magnetic beads | life technologies | 11201D | These Dynabeads are superparamagnetic beads with affinity purified polyclonal sheep anti-mouse IgG covalently bound to the bead surface. Store at 4 °C. |
Magnetic beads | life technologies | 11203D | These Dynabeads are superparamagnetic beads with affinity purified polyclonal sheep anti-rabbit IgG covalently bound to the bead surface. Store at 4 °C. |
Magnetic beads | life technologies | 10001D | These Dynabeads are superparamagnetic beads with recombinant protein A covalently bound to the bead surface. Store at 4 °C. |
Magnetic beads | life technologies | 10003D | These Dynabeads are superparamagnetic beads with recombinant protein G covalently bound to the bead surface. Store at 4 °C. |
Magnetic rack | life technologies | 12321D | DynaMag-2 magnet |
MEME | Bailey et al. | non-commercial | http://meme.nbcr.net/meme/ |
Na3VO4 | New England BioLabs | P0758 | Sodium orthovanadate (100 mM) is a commonly used general inhibitor for protein phosphotyrosyl phosphatases. Store at -20 °C. |
NaF | New England BioLabs | P0759 | Sodium fluoride (500 mM) is commonly used as general inhibitor of phosphoseryl and phosphothreonyl phosphatases. Store at -20 °C. |
NGS machine | Illumina | SY-301-1301 | Genome Analyzer IIx |
NGS machine (high performance) | Illumina | SY-401-2501 | HiSeq |
Normal serum (antibody control) | Santa Cruz Biotechnology | sc-2028 | Use as control for goat polyclonal IgG antibodies in ChIP-qPCR experiments. Store at 4 °C. |
Normal serum (antibody control) | Santa Cruz Biotechnology | sc-2025 | Use as control for mouse polyclonal IgG antibodies in ChIP-qPCR experiments. Store at 4 °C. |
Normal serum (antibody control) | Santa Cruz Biotechnology | sc-2027 | Use as control for rabbit polyclonal IgG antibodies in ChIP-qPCR experiments. Store at 4 °C. |
Nucleic acid staining solution | iNtRON | 21141 | Use RedSafe nucleic acid staining solution at 1:50,000. Store at room temperature. |
Orange G | Sigma | O3756-25G | 1-Phenylazo-2-naphthol-6,8-disulfonic acid disodium salt. Store at 4 °C. |
PCR primers | e.g., IDT or Sigma | NA | 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 µM each. Use 5 µl in a 50 µl PCR reaction. Store at -20 °C. |
MinElute PCR purification kit | Qiagen | 28006 | for purification of ChIP-qPCR and shearing test samples. Store MinElute spin columns at 4 °C, all other buffers and collection tubes at room temperature. |
Phenol:chloroform:isoamyl alcohol (25:24:1, pH 7.9) | life technologies | AM9730 | 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 °C. CAUTION: phenol:chloroform:isoamyl alcohol is corrosive, highly toxic and combustible. |
Primer3 (software) | Steve Rozen & Helen Skaletsky | non-commercial | http://biotools.umassmed.edu/bioapps/primer3_www.cgi |
Protease inhibitor tablets | Roche | 11836170001 | cOmplete, Mini, EDTA-free. Use 1 tablet per 10 ml. Store at 4 °C. |
Protease inhibitor tablets | Roche | 11873580001 | cOmplete, EDTA-free. Use 1 tablet per 50 ml. Store at 4 °C. |
Proteinase K | life technologies | AM2548 | proteinase K solution (20 µg/µl). Store at -20 °C. |
RNase A | life technologies | 12091-039 | RNase A (20 µg/µl). Store at room temperature. |
Rotator | Stuart | SB3 | Rotator SB3 |
SAMtools (software) | Li et al. | non-commercial | http://samtools.sourceforge.neta |
Solid phase reversible immobilisation beads | Beckman Coulter | A63882 | The Agencourt AMPure XP beads are used to minimise adaptor dimer contamination in ChIP-Seq libraries. Store at 4 °C. |
Sonicator 3000 | Misonix/Qsonica | NA | Newer models are now available. Q125, Q500 or Q700 are all suitable for shearing crosslinked chromatin. |
Sound enclosure | Misonix/Qsonica | NA | optional: follow the manufacturer's recommendation to obtain the correct sound enclosure. |
Thermomixer | eppendorf | 22670000 | Thermomixer for 24 x 1.5 mL tubes. Precise temperature control from 4 °C above room temperature to 99 °C. |