La cuestión de cómo los reguladores y los estados de la cromatina cromatina afecta al genoma in vivo es fundamental para nuestra comprensión de cómo los primeros se toman las decisiones del destino celular en el embrión en desarrollo. Chip-Sec-el método más popular para investigar las características de la cromatina en un nivel se describe aquí mundial para los embriones de Xenopus.
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.
Nuestro protocolo describe cómo realizar y analizar los perfiles de la cromatina en todo el genoma a partir de embriones de Xenopus. Cubre cada paso de las proteínas de reticulación a loci endógenos in vivo para procesar millones de lecturas que representa sitios genómicas enriquecidas en silico. Dado el creciente número de borradores del genoma están disponibles, este protocolo debe ser aplicable a otros organismos modelo y no modelo. La sección experimental más importante, lo que diferencia a este protocolo aparte del trabajo previo 8,31,33,34, es el procedimiento posterior a la fijación para extraer núcleos reticulados. Se facilita la solubilización de la cromatina eficiente y de cizallamiento y fácil ampliación de la escala. Junto con la mejora de la eficiencia de la preparación de la biblioteca este protocolo permite la construcción de bibliotecas-chip Sec de alta complejidad de la mitad a dos millones de células que expresan el epítopo asociada a la cromatina de interés. Para los experimentos chip-qPCR, algunos diez mil de estas células son normalmente suficientepara comprobar para el enriquecimiento de ADN en unos seis loci genómicos diferentes. Estos números son estimaciones conservadoras, pero puede variar dependiendo del nivel de expresión de la proteína, la calidad del anticuerpo, la eficiencia, y la accesibilidad epítopo de reticulación. Como una guía, un solo embrión de Xenopus contiene alrededor de 4.000 células en la etapa de mediados de blástula (8,5 después de Nieuwkoop y Faber 29), 40.000 células en la última etapa de gástrula (12) y 100.000 células en la fase tailbud temprana (20).
La hora exacta de fijación para la inmunoprecipitación eficaz necesita ser determinado empíricamente por chip-qPCR (sección 10). En general, se requieren tiempos de fijación más largos si el experimento implica X. laevis embriones, primeras etapas de desarrollo, y propiedades de unión de ADN débiles (o indirectos). Sin embargo, no se recomienda la fijación de Xenopus embriones de más de 40 minutos, o el procesamiento de más embriones que los indicados (sección 3), cuya esquila cromatina se vuelve menos eficiente. Es importante noutilizar cualquier glicina después de la fijación ya que este paso común para saciar el formaldehído puede hacer extracción nuclear a partir de embriones de yema rica muy difíciles. Actualmente, no se conoce la razón de esto. Es concebible que el aducto de formaldehído glicina reacciona adicionalmente con grupos amino o residuos de arginina 35 N-terminal.
El anticuerpo es clave para cualquier experimento de chip y controles suficientes necesita ser llevado a cabo para mostrar su especificidad para el epítopo de interés (ver directrices por Landt et al. 36). Si ningún anticuerpo chip-grado se encuentra disponible, la introducción de proteínas de fusión etiquetadas con epítopo correspondiente puede ser una alternativa legítima como estas proteínas pueden ocupar sitios de unión 37 endógeno. En este caso, los embriones no inyectados son los mejores para usar como un control negativo en lugar de un chip con suero no específico. Esta estrategia también se puede aplicar si la proteína de interés se expresa en niveles bajos resultantes en el pobre recuperación de enriADN ched.
Como para la fabricación de bibliotecas-Chip SEQ, debido a la baja cantidad de ADN en uso, se recomienda optar por los procedimientos que reducen el número de pasos de limpieza y para reacciones combinan para mantener cualquier pérdida de ADN en un mínimo. Los adaptadores y cebadores tienen que ser compatibles con la secuenciación multiplex y la plataforma de NGS (véase la Tabla de Materiales Específicos / Equipo). Si se utiliza adaptadores Y (que contiene los brazos de una sola hebra de largo), es crítico para pre-amplificar la biblioteca con tres a cinco rondas de PCR antes de insertos de ADN de tamaño-seleccionar (por ejemplo., De 100 a 300 pb) mediante electroforesis en gel. Extremos de cadena simple causan fragmentos de ADN que migran de forma heterogénea. Trial se ejecuta con diferentes cantidades de ADN de entrada (por ejemplo, 0,1, 0,5, 1, 2, 5, 10 y 20 ng) se recomienda para determinar el número total de ciclos de PCR (menos de o igual a 18 ciclos) requerida para hacer un tamaño -seleccionado biblioteca de 100 a 200 ng. La reducción del número de ciclos de PCR hace que la secuenciación de redulee menos probable ndant. En fase sólida perlas de inmovilización reversibles son buena limpieza de reactivos para recuperar de manera eficiente el ADN de interés y fiable extraer cualquier adaptador y dímeros libres de la ligadura y reacciones de PCR.
En términos de número, tipo y duración de la lee, alrededor de 20 y 30 millones de fin de lecturas simples de 36 pb es suficiente para la mayoría de los experimentos-chip Sec para cubrir todo el genoma Xenopus con la profundidad suficiente. Las máquinas NGS más prevalentes son rutinariamente capaz de satisfacer estos criterios. Sin embargo, puede ser beneficioso para aumentar el número de lecturas, si se espera que un amplias distribuciones de lee, como se observa con las modificaciones de histonas, en lugar de picos agudos. Para muchos experimentos-chip Sec, 4-5 bibliotecas diferente indexados se unen y se secuenciaron en el carril celular uno de flujo usando una máquina de NGS alto rendimiento. A veces también es aconsejable extender la longitud de lectura y la secuencia de los dos extremos de la plantilla de ADN (de extremo emparejado) para aumentar mapeabilidad wanalizar gallina cromatina dentro de las regiones genómicas repetitivas.
Este protocolo se ha aplicado con éxito a una amplia variedad de características de la cromatina, tales como factores de transcripción, mediadores de señalización y las modificaciones de histonas post-traduccionales. Sin embargo, los embriones adquieren un creciente grado de heterogeneidad celular a medida que desarrollan y los perfiles de la cromatina se vuelven más difíciles de interpretar. Pasos prometedores se han hecho en Arabidopsis y Drosophila a los paisajes de la cromatina perfil específicamente mediante la extracción de tejido de tipo específico núcleos celulares 38,39. Nuestro protocolo incluye una etapa de extracción nuclear, que podría allanar el camino para que los tejidos específicos de chip-Sec en otros embriones.
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. |