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.

우리의 프로토콜은하고 아프리카 발톱 개구리의 배아에서 게놈 전체 염색질 프로파일을 분석하는 방법에 대해 설명합니다. 그것은 실리에 풍부한 게놈 사이트를 나타내는 읽기의 수백만을 처리하는 생체 내에서 내인성 유전자 좌에 가교 단백질에서 모든 단계를 다루고 있습니다. 게놈 초안의 증가 번호를 사용할 수 있기 때문에,이 프로토콜은 다른 모델과 비 모델 생물에 적용해야합니다. 이전의 연구 8,31,33,34에서 떨어져이 프로토콜을 설정하는 가장 중요한 실험 부분은, 가교 핵을 추출 후 고정 절차입니다. 그것은 효율적인 염색질 용해 화 및 전단 쉽게 업 스케일링을 용이하게한다. 함께 도서관 준비의 개선 된 효율성이 프로토콜은 반에서 관심있는 염색질 관련 항원을 발현하는 이백만 세포에 높은 복잡성 칩-SEQ 도서관의 건축을 할 수 있습니다. 칩 qPCR의 실험의 경우, 이들 세포의 몇 만 정상적으로 충분아마도 여섯 별개의 게놈 유전자 좌에서 DNA 농축을 확인합니다. 이러한 숫자는 보수적이지만, 단백질 발현 수준, 항체의 품질에 따라 효율, 에피토프의 접근성을 가교 달라질 수있다. 가이드로, 단일 Xenopus의 배아 중반 포배 단계 (Nieuwkoop 및 페이버 (29) 후 8.5)에서 약 4,000 세포를 포함, 후반 낭배 단계 (12)에서 40,000 세포와 초기 tailbud 단계 (20)에서 10 만 셀. 효율적인 면역에 대한 정확한 고정 시간은 칩 qPCR에 (10 항)에 의해 경험적으로 결정해야합니다. 실험이 X를 포함하는 경우 일반적으로 더 이상 고정 시간이 필요합니다 배아, 초기 발달 단계, 약한 (또는 간접) DNA 결합 특성을 laevis의. 그러나, 염색질 전단 효율성이 떨어질대로, 40 분보다 긴 아프리카 발톱 개구리 배아를 고정, 또는 표시 (섹션 3)보다 더 많은 배아를 처리하지 않는 것이 좋습니다. 그것은 중요하지매우 어려운 노른자 풍부한 배아에서 핵 추출을 할 수 포름 알데히드를 담금질이 일반적인 단계로 고정 후 어떤 글리신를 사용합니다. 현재, 그 이유는 알려져 있지 않다. 또한 포름 알데히드 부가 물 상기 글리신 아미노 기 또는 아르기닌 잔기 35 N 말단과 반응하여 생각할 수있다. 항체는 칩 실험의 핵심입니다 충분한 컨트롤 (Landt 등. (36) 가이드 라인을 참조) 관심의 항원에 대한 특이성을 보여주기 위해 수행 될 필요가있다. 더 칩 등급 항체가없는 경우이 단백질이 차지하는 결합 부위 내인성 수 37, 에피토프 태그 융합 단백질 대응 소개 합법적 대안이 될 수있다. 이 경우, uninjected 배아는 음성 대조군보다는 비특이적 혈 칩으로 사용하는 것이 최선이다. 관심의 단백질이 enri의 가난한 복구의 결과로 낮은 수준에서 표현되는 경우,이 전략은 적용 할 수있다CHED DNA. 왜냐하면 사용중인 DNA의 소량 칩-SEQ 라이브러리 제조용으로서,이 세정 공정 수를 줄이고 최소한 DNA의 손실을 유지하는 반응을 조합하는 방법을 선택하는 것을 권장한다. 어댑터와 프라이머 (특정 재료 / 장비의 표 참조) 멀티 플렉스 시퀀싱 및 NGS 플랫폼과 호환 될 수 있도록해야합니다. (긴 단일 가닥 무기를 포함) Y-어댑터를 사용하는 경우, 그것은 크기 선택 DNA 삽입하기 전에 PCR의 3-5 라운드 사전 증폭 라이브러리에 중요하다 (예., 100 내지 300 bp) 겔 전기 영동에 의해. 단일 가닥 말단 DNA 단편이 균질하게 이동. 시험은 PCR 사이클의 총 수를 결정하도록 권장 입력 DNA (예를 들면, 0.1, 0.5, 1, 2, 5, 10, 20 NG)의 다양한 양으로 실행 (이하 또는 18 사이클 같음) 크기를 만드는 데 필요한 100-200 NG의 - 선정 된 라이브러리입니다. PCR 사이클의 수를 줄이면 redu의 시퀀싱을 렌더링ndant은 덜 읽습니다. 고상 가역적 고정화 비즈 효율적으로 관심있는 DNA를 복구하고 안정적​​으로 결찰 및 PCR 반응이없는 임의의 어댑터 및 이량 체를 제거하는 시약을 정리 좋다. 수, 유형 및 길이의 측면에서 약 20-30000000 단일 엔드 36 bp의 충분한 깊이와 전체 Xenopus의 게놈을 커버하는 대부분의 칩 서열 번호 실험에 충분하다의 읽기, 읽기의. 가장 널리 NGS 기계는 이러한 기준을 충족 일상적으로 할 수있다. 그러나, 판독의 광범위한 분포가 예상되는 경우의 판독 샤프한 피크보다 오히려, 히스톤 변형 관찰로, 수를 늘리는 것이 유리할 수있다. 다수의 칩 - 서열 번호 실험을 위해, 4-5 다르게 인덱싱 라이브러리 풀링 할 수 있고, 고성능 NGS 기계를 사용하여 하나 이상의 유동 세포 차선 서열화. 때때로 mappability w을 증가 판독 길이 및 서열 DNA 템플릿의 양단 (페어링 엔드)를 연장하는 것이 바람직하다반복적 인 게놈 영역 내에서 염색질 암탉 분석입니다. 이 프로토콜은 전사 인자 시그널링 매개체 번역 후 변형으로 히스톤 염색질 다양한 기능에 성공적으로 적용되었다. 그들이 개발하고 염색질 프로파일을 해석하기 어렵게 될 그러나, 배아 세포 이질성의 증가 학위를 취득. 유망 단계는 세포 유형 특정 핵 (38, 39)를 추출하여 조직 특별히 프로필 염색질 풍경에 애기 장대와 초파리되었습니다. 우리의 프로토콜은 다른 배아 조직 특이 칩 서열 번호에 대한 방법을 포장 할 수있는 핵 추출 단계를 포함한다.

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.

프로토콜은 잘 실행되는 경우 여기 제시된 동등한 결과가 예상되는 사용 및 항체 칩 품질 등급 (설명 참조)이다. 이 프로토콜은 포름 알데히드 고정 아프리카 발톱 개구리의 배아에서 핵의 추출 및 초음파에 의한 염색질의 효율적인 전단 (그림 1A-C)를 할 수 있습니다. 전단 염색질은 주로 100 내지 1000 염기쌍의 범위 및 BP (도 1C) 300 내지 500 피킹 DNA 단편의 비대칭 분포를 나타낸다. 면역 DNA의 최소 50 페이지가 성공적으로 비슷한 크기의 DNA를 삽입 (그림 2A)와 인덱스 쌍 엔드 칩-SEQ 라이브러리를 만들기 위해 필요합니다. 도서관은 약 12​​0 bp의 electropherogram에서 볼 수 있습니다 어댑터 이량 체의 크게 결여해야한다. 시퀀싱 합성에 의한시 읽기 사전 처리는 게놈 (그림 2B, C)에 매핑됩니다. 성공적인 실험과에서 <em&gt; X. tropicalis 배아는 하나의 엔드의 일반적 50-70%는 최대한 두 불일치와 7.1의 게놈 어셈블리에 고유하게 매핑 할 수 있습니다 (40) 혈압 읽습니다. 입력 게놈에 걸쳐 매우 균일하게 정렬 읽는 동안, 칩의 정렬은 관심 염색질 기능의 측면 가닥 별 부화에 결과를 읽습니다. 조각을 모두 5 &#39;말단 (그림 2C) 25 서열 때문입니다. 평균 단편 크기에 방향을 읽는 정렬을 확장하는 것은 같은 전사 인자 바인딩 이벤트와 같은 하나의 크로 마틴 기능에 대한 정확한 프로파일을 생성합니다. IGV 또는 기타 호환 게놈 브라우저 시각화 할 때 이러한 DNA의 점유는 피크로 나타납니다. MACS 등 피크 발신자는 이들 피크 (도 3a)의 위치를 결정하기 위해 사용된다. 수십 결합 부위의 수천이 방법은 X에서 결정되었습니다 tropicalis 게놈 같은 VegT (26) T-상자 전사 인자에 대한. 칩 qPCR에의 체험관사항 칩-SEQ (그림 3B)에 의해 발견 된 지역 농축을 확인해야합니다. 칩 서열 번호 실험 염색질 기능의 게놈 전체의 특성을 탐구 할 수 있습니다. 예를 들어, 전사 개시 및 종결 부위로 게놈 요소를 판독 분포를 산출하는 임의의 유전자 주위 공간 결합 선호도 (도 3c)를 강조 할 수있다. 마찬가지로, 피크 위치에서 읽기 분포의 히트 맵은 게놈 전체 규모 (그림 3D)에서 다른 염색질 기능을 비교하는 데 사용됩니다. 특정 전사 인자는 잠재적 인 공동 요인 (그림 3E)의 공동 풍부한 주제를 포함하여 이러한 종류의 정보를 검색 할 수 있습니다 게놈 DNA 기본 피크의 DNA 서열 특히. 드 노보 모티브 분석을 결합한다. 표적 유전자의 대다수는 DNA의 하부에 점유보다는 더 높은 레벨 (도 3f)를 도시한다. 이 규모없는 기능은 그럴 사이에서 매우 일반적인 것 같다anscription 인자와 표적 유전자의 작은 부분을 직접 생물학적 관련성 27,28으로 조절되는 것을 제시한다. 이러한 추가 Xenopus의 배아 (그림 3G)에서 염색질 기능의 생물학적 기능에 대한 통찰력을 공개 할 수있다 표적 유전자의 발현 차이 등 풍부한 GO의 조건이나 다른 속성의 분석. <img alt="그림 1" src="/files/ftp_upload/52535/52535fig1highres.jpg" /> 아프리카 발톱 개구리의 배아 1. 염색질 면역 침전 과정을 그림. (A) 배아 포름 알데히드 고정 공유 바인드 (가교)에 관심 게놈 DNA와 관련된 단백질의 발달 단계에있다. 핵 추출 (B)시, 가교 염색질 바인딩 게놈 DNA 또는 측면에서는 DN을 최소화하여 염색질 변형 사이트를 좁힐 조각난시퀀스 (C). 그 후, 염색질 조각은이자 (D)의 에피토프를 포함하는 사람들을 풍요롭게 칩 수준의 항체와 면역 있습니다. 공동 면역 DNA가 단백질을 제거하고 NGS (그림 2)의 칩 조각 라이브러리를 작성하기 전에 (E)를 정제한다. <a href="https://www.jove.com/files/ftp_upload/52535/52535fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/52535/52535fig2highres.jpg" /> 그림 2. 칩 서열 번호 라이브러리 준비, 시퀀싱 합성에 의한, 매핑 및 피크를 호출합니다. (A) electropherogram는 250 bp의 450의 DNA 템플릿과 좋은 칩 서열 번호 라이브러리를 표시합니다. 이러한 템플릿은 보편적 (58 BP)에 의해 형벌 관심의 DNA 삽입 및 인덱스 (63 BP) 어댑터를 수반한다. (B) </동일한 템플릿을 포함하는 각 클러스터와 클러스터의 강한&gt; 수백만, 가역, 독특한 형광과 동일한 종료 속성을 가진 네 개의 뉴클레오티드의 존재에 기초하여 시퀀스 기본이다. 형광 이미지 읽기에 궁극적으로 조립 해당 기지를, 전화를 실시간으로 처리됩니다. (C)는 만 유일하게 아프리카 발톱 개구리의 게놈지도가 유지되는 것을 읽습니다. 모든 단편은 5 &#39;말단으로부터 순서가되기 때문에, 칩의 매핑은 관심 염색질 기능의 측면 가닥 특정 피크의 결과를 판독한다. 이에 따라, 피크 호출자가 정확하게 크로 마틴 기능을 현지화 평균 조각 길이로 읽어 면역에서 유래하고 확장 농축을 감지합니다. <a href="https://www.jove.com/files/ftp_upload/52535/52535fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img aLT = &quot;그림 3&quot;SRC = &quot;/ 파일 / ftp_upload / 52535 / 52535fig3highres.jpg&quot;/&gt; 그림 3. 접합체의 T-상자 전사 인자 VegT (zVegT). 모두 여기에 표시된 수가 10 만명 정규화 읽어 고유 매핑과 비 중복이 읽습니다. (A) 발췌에 의한 후 염기 서열 분석 및 데이터 시각화의 예 zVegT의 게놈 전체 프로필 X에서 바인딩 (Nieuwkoop 및 페이버 (29) 후 12.5로 무대 11) tropicalis 낭배 배아. 각 피크, 확장 된 읽기의 더미 업, 하나의 결합 부위를 나타냅니다. 이들 피크는 1 % 미만의 오류 발견 레이트 (FDR)와 MACS2 의해 호출된다. 각 mesp 유전자 바인딩 매우 인접 상류 zVegT를 보여줍니다,하지만 mespa 및 mespb는 그 단계 (RNA-SEQ 데이터 30)에 의해 표현된다. (B) zVegT의 DNA 점유율 수준 여러 유전자 ​​좌에서 칩 qPCR에 의해 결정 (비 포함 KB 상류 β 액틴의 -bound 지역 0.5) 확인 합니칩 서열 번호에 의해 발견 된 특정 농축을 RM은. (A)에서 (빨간색 막대)라고 피크 mespa에 대한 결과를 비교한다. DNA 점유 레벨은 모두 입력의 비율, VegT 항체 (IgG의 이소 타입의 토끼 폴리 클로 날) 및 항체 제어 (정상 토끼의 IgG)와 칩 (Chip) 칩으로 보여집니다. 오차 막대는 두 개의 생물학적 반복 실험의 표준 편차를 반영한다. (C) Metagene 분석 주위에 다른 게놈 영역 및 유전자 기관 내 프로모터 상대에 바인딩 우선 zVegT을 (태그 25 bp의 이상 비닝)를 보여줍니다. (D)는 히트 맵은 K-평균을 보여줍니다 클러스터 (K = 5) DNA 점유율 수준 낭배 단계에서 모든 zVegT 바인딩 지역에 zVegT과 된 Smad2 /의 Smad3 (칩 서열 번호 데이터 31) 상대의 (태그는 25 bp의 이상 비닝). 히트 맵은 드 노보 모티브 분석 zVegT-의 38 %에 결합 모티프 정식 T-상자 전사 인자를 발견 2 기반 BP 당 5 태그를 중심으로. (E) 로그입니다기본 모티브 스코어 배경 서열에서 5 %의 비율로 발견 정규화 경우 결합 영역. 정식 된 Smad2 / Smad3이 결합 모티프가 거의 농축되지 않은 반면, 밀도지도, zVegT 결합 부위의 중앙에 T-상자 모티브 가장 높은 농축을 보여줍니다. (F) 히스토그램은 각 표적 유전자에 대해 계산된다 zVegT의 DNA 점유율 수준을 보여줍니다 [-] 모든 피크 (+/- 200 BP) 5킬로바이트 상류 사이에서. 전사 시작 사이트를 대응하고 1킬로바이트 하류 [+] (G) 가장 높은 DNA 점유율 내 -5 킬로바이트 수준과 1킬로바이트 아르와 최고 300 유전자 초기 배아 발달의 생물학적 과정에 대한 풍부. 이 용어가 zVegT의 추정 함수와 함께 줄에 이동합니다. FDR은 양측 피셔의 정확한 테스트를 기반으로 여러 테스트를 위해 수정됩니다. <a href="https://www.jove.com/files/ftp_upload/52535/52535fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</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

방법 염색질 규제 및 염색질 상태의 문제는 생체 내에서 유전자가 세포 운명 결정이 배아에서 어떻게 만들어 지는지 초기에 대한 우리의 이해의 열쇠입니다 영향을 미칩니다. 칩 서열 번호 - 글로벌 수준입니다 Xenopus의 배아 여기 설명에서 크로 마틴의 기능을 조사하기 가장 인기있는 방법.

염색질 레귤레이터 및 미국에서의 게놈 전체의 스냅 샷<em&gt; 아프리카 발톱 개구리</em칩 서열 번호&gt; 태아

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

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Developmental Biology

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

12.1K 조회수.  MRC National Institute for Medical Research.  방법 염색질 규제 및 염색질 상태의 문제는 생체 내에서 유전자가 세포 운명 결정이 배아에서 어떻게 만들어 지는지 초기에 대한 우리의 이해의 열쇠입니다 영향을 미칩니다. 칩 서열 번호 - 글로벌 수준입니다 Xenopus의 배아 여기 설명에서 크로 마틴의 기능을 조사하기 가장 인기있는 방법. 

비디오: 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

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

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

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

조작 및<em&gt; 체외</em의&gt; 성숙<em&gt; 아프리카 발톱 개구리 laevis의</em세포질 내 정자 주입에 이어&gt; 난 모세포, 배아 발달을 연구하는

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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Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, the ability to determine an embryo&#39;s sex is necessary in many experiments particularly regarding early development. The present protocol demonstrates an inexpensive, rapid and non-toxic preparation of pig genomic DNA for use with PCR. Day 30 embryos must be humanely collected according to the guidelines established by Institutional Animal Policy and Welfare Committees for the present protocol. The preparation of the whole embryo for this PCR based sexing technique simply involves grinding the frozen embryo to a fine powder using a pre-chilled mortar and pestle. PCR-quality DNA is released from a small amount of embryo powder by applying a hot incubation in an alkaline lysis reagent. Next, the DNA solution is mixed with neutralization buffer and used directly for PCR. Two primer pairs are generated to detect specific sex determining region of the Y- chromosome (SRY) and ZFX region of the X- chromosome with high accuracy and specificity. The same protocol can be applied to other elongated embryos (Day 10 to Day 14) earlier than Day 30. Also, this protocol can be carried with 96-welled plates when screening a large number of embryos, making it feasible for automation and high-throughput sex typing.

This protocol describes an accurate, inexpensive, rapid and non-toxic method to determine the sex of Day 30 porcine embryo using PCR method after grinding an embryo into powder without phenol chloroform extraction and DNA column purification.

PCR에 의한 날 (30) 돼지 배아의 정확하고 페놀 무료 DNA Sexing

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, ...

Technique to Target Microinjection to the Developing Xenopus Kidney

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 embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

개발에 미세 주입을 대상으로 기술<em&gt; Xenopus의</em&gt; 신장

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 ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

Blastulae 개발의 외과 융합에 의해 Parabiotic 제브라 피쉬 배아의 생성

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

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

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

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

의 배아 혈관 신생에 대한 시각화 및 정량 분석<em&gt; Xenopus tropicalis</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

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 zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

살아있는 개구리와 Zebrafish 태아의 저산소증 유도

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 ...

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-Seq와 칩 Seq: Chromatin 관련 된 RNAs 및 배아 줄기 세포에 단백질 DNA 상호 작용을 식별 하는 방법

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

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

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

노드 및 스캐닝 전자 현미경 검사 법, 면역 형광 산 전체를 사용 하 여 Gastrulating 마우스 배아에서 Notochordal 접시를 시각화

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

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

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

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

제노푸스 laevis 배아에서 눈부안으로 DNA의 미세 주입 및 GFP의 이미징 그대로 광학 축색 아버를 표현, 살아있는 제노푸스 올챙이

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