עבודה זו נתמכה על ידי ויין סטייט, Karmanos סרטן אוניברסיטאי, מענק מן הלאומי ללב, ריאות ודם המכון (1K22HL126842-01A1) הוענק B.L.K. עבודה זו מנוצל ויין המדינה אוניברסיטת גבוהה ביצועי מחשוב ברשת עבור משאבים חישובית (< https://www.grid.wayne.edu/>). אנו מודים ג'יג'י Kurup על סיוע עם ניתוח נתונים CARIP-Seq.
התרומות של המחברים:
B.L.K. היגו בשיטת CARIP-Seq, תוכנן ביצעו ניסויים שבב-Seq ו- CARIP-Seq, ניתח את הנתונים רצף ו ניסח את כתב היד. כל המחברים לקרוא, אישר את הגרסה הסופית של כתב היד הזה.

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X., Liu, C., Zhao, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+self-renewal+and+accelerated+reprogramming+in+the+absence+of+Kdm5b.">Extended self-renewal and accelerated reprogramming in the absence of Kdm5b.</a> Mol Cell Biol. 33, 4793-4810 (2013).</li><li>Kidder, B. 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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mRNA-bound+proteome+and+its+global+occupancy+profile+on+protein-coding+transcripts.">The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts.</a> Mol Cell. 46 (5), 674-690 (2012).</li><li>Castello, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+RNA+biology+from+an+atlas+of+mammalian+mRNA-binding+proteins.">Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.</a> Cell. 149 (6), 1393-1406 (2012).</li><li>Hudson, W. H., Ortlund, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure,+function+and+evolution+of+proteins+that+bind+DNA+and+RNA.">The structure, function and evolution of proteins that bind DNA and RNA.</a> Nat Rev Mol Cell Bio. 15 (11), 749-760 (2014).</li><li>Khalil, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Many+human+large+intergenic+noncoding+RNAs+associate+with+chromatin-modifying+complexes+and+affect+gene+expression.">Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression.</a> P Natl Acad Sci USA. 106 (28), 11667-11672 (2009).</li><li>Hendrickson, D. G., Kelley, D. R., Tenen, D., Bernstein, B., Rinn, J. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+long+noncoding+RNAs+in+epigenetic+regulation.">Structure and function of long noncoding RNAs in epigenetic regulation.</a> Nat Struct Mol Bio. 20 (3), 300-307 (2013).</li><li>Chu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+discovery+of+Xist+RNA+binding+proteins.">Systematic discovery of Xist RNA binding proteins.</a> Cell. 161 (2), 404-416 (2015).</li><li>Mak, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivation+of+the+paternal+X+chromosome+in+early+mouse+embryos.">Reactivation of the paternal X chromosome in early mouse embryos.</a> Science. 303 (5658), 666-669 (2004).</li><li>Garfield, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+primary+mouse+embryonic+fibroblast+(PMEF)+cultures.">Derivation of primary mouse embryonic fibroblast (PMEF) cultures.</a> Methods Mol Biol. 633, 19-27 (2010).</li><li>Conner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+embryo+fibroblast+(MEF)+feeder+cell+preparation.">Mouse embryo fibroblast (MEF) feeder cell preparation.</a> Curr Protoc Mol Biol. , (2001).</li><li>Ying, Q. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ground+state+of+embryonic+stem+cell+self-renewal.">The ground state of embryonic stem cell self-renewal.</a> Nature. 453 (7194), 519-523 (2008).</li><li>Langmead, B., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+gapped-read+alignment+with+Bowtie+2.">Fast gapped-read alignment with Bowtie 2.</a> Nat Methods. 9 (4), 357-359 (2012).</li><li>Zang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clustering+approach+for+identification+of+enriched+domains+from+histone+modification+ChIP-Seq+data.">A clustering approach for identification of enriched domains from histone modification ChIP-Seq data.</a> Bioinformatics. 25 (15), 1952-1958 (2009).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model-based+analysis+of+ChIP-Seq+(MACS).">Model-based analysis of ChIP-Seq (MACS).</a> Genome Biol. 9 (9), R137 (2008).</li><li>Liu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+model-based+Analysis+of+ChIP-Seq+(MACS)+to+analyze+short+reads+generated+by+sequencing+protein-DNA+interactions+in+embryonic+stem+cells.">Use model-based Analysis of ChIP-Seq (MACS) to analyze short reads generated by sequencing protein-DNA interactions in embryonic stem cells.</a> Methods Mol Biol. 1150, 81-95 (2014).</li><li>Kidder, B. L., Hu, G., Zhao, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-Seq:+technical+considerations+for+obtaining+high-quality+data.">ChIP-Seq: technical considerations for obtaining high-quality data.</a> Nat Immunol. 12 (10), 918-922 (2011).</li><li>Quail, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large+genome+center's+improvements+to+the+Illumina+sequencing+system.">A large genome center's improvements to the Illumina sequencing system.</a> Nat Methods. 5 (12), 1005-1010 (2008).</li><li>Schmidl, C., Rendeiro, A. F., Sheffield, N. 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המחברים מצהירים ללא ניגוד אינטרסים.

שבב-Seq היא שיטה שימושית כדי להעריך את המיקום של אינטראקציות חלבון העולמי-DNA (למשל., גורמי שעתוק/שינוי שינויים אנזימים/היסטון ודנ א היסטון) תאים ES, בעוד פרוטוקול CARIP-Seq פיתח הוא שימושי חוקר את השיוך ברמת הגנום של RNAs המרכיבים כרומטין. שבב-Seq הוא כלי בסיסי המשמש כדי להעריך נופים epigenetic של תאים ...

החלטות גורל תאי גזע עובריים (ES) מוסדרים על ידי תקשורת בין אותות חוץ-תאית וכן שורה של גנים ברמת השעתוק-וסתים, כולל היסטון מגבילים, תרגום שלאחר שינוי של היסטון זנבות. אינטראקציות אלה להקל על נגישות כרומטין, אריזה של כרומטין לתוך אחד של שתי מדינות: euchromatin, אשר פתוח ופעיל transcriptionally, ואת הטרוכרומטין, אשר הוא קומפקטי וקל בדרך כלל transcriptionally לא פעיל. גורמי שעתוק DNA רצף ספציפי איגוד הזיקות, עמית מכפילי epigenetic עם אזורים תהיה להשתתף בשליטה על ביטוי גנים. הדור הבא רצפי שיטות, כולל צ'יפ-Seq1, היה אינסטרומנטלי מיפוי הגנום כולו רשתות תעתיק מהותי עבור ES תא התחדשות עצמית, pluripotency2,3,4 5, ,6. יתר על כן, בעוד immunopreciation RNA ואחריו הדור הבא רצף (RIP-Seq)7 ההערכות של אינטראקציות חלבון ה-RNA מציע כי ה-DNA מחייבת חלבונים אינטראקציה עם RNAs להסדיר אירועים תעתיק7, 8 , 9 , 10 , 11 , 12, מחקרים מעטים חקרו לוקליזציה ברמת הגנום של RNAs המשויך כרומטין12או גלובלי האינטראקציות בין שינויים RNA והיסטון. זמן אי קידוד RNAs (lncRNAs) הם קבוצה אחת של RNAs אשר נמצאו להסדיר את הפעילות של חלבונים הקשורים כרומטין13,14,15. לדוגמה, Xist הוא lncRNA המווסת, בתאים בתרבית של נקבה, איון של כרומוזום X אחד, דרך הגיוס של epigenetic repressors16,17. אולם, מלוא הספקטרום של RNAs הקשורים עם כרומטין הוא אינו מודע לקיומם. כאן, אנו מתארים את פרוטוקול הרומן, הקשורים כרומטין RNA immunoprecipitation (CARIP) ואחריו הדור הבא רצפי (CARIP-Seq), לזיהוי הקשורים כרומטין RNAs על בסיס הגנום כולו, תאים ES, כולל הכנה ספריה עבור הדור הבא רצפי, צ'יפ-Seq למפות תפוסת הכללית של שינויים היסטון, גורמי שעתוק, מכפילי epigenetic. שלא כמו שאר שיטות RIP-Seq7, CARIP-Seq כולל שלבים crosslinking ו sonication, המאפשרים זיהוי ישיר RNAs הקשורים עם כרומטין. יחד, צ'יפ-Seq הוא כלי רב עוצמה לזיהוי אינטראקציות חלבון-DNA הגנום כולו, בזמן CARIP-Seq היא שיטה חזקה סקר RNAs הקשורים רכיבים כרומטין.

<table><tbody><tr><td>Mouse leukemia inhibitory factor (ESGRO LIF)</td><td>EMD Millipore</td><td></td><td>10&lt;sup&gt;6 &lt;/sup&gt;or 10&lt;sup&gt;7 &lt;/sup&gt;units</td></tr><tr><td>GSK3&amp;beta; inhibitor CHIR99021 (GSK3i)</td><td>Stemgent</td><td></td><td>Solvent: DMSO 10mM</td></tr><tr><td>MEK inhibitor PD0325901 (MEKi)</td><td>Stemgent</td><td></td><td>Solvent: DMSO 10mM</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM), high glucose</td><td>Invitrogen</td><td>11965092</td><td></td></tr><tr><td>PBS (phosphate buffered saline)</td><td>Invitrogen</td><td>10010031</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Gibco</td><td>25200056</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>2-Mercaptoethanol (50 mM)</td><td>Gibco</td><td>31350010</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100X)</td><td>Gibco</td><td>11140076</td><td></td></tr><tr><td>EmbryoMax ES Cell Qualified Fetal Bovine Serum, 500 ml</td><td>EMD Millipore</td><td>ES-009-B</td><td></td></tr><tr><td>EmbryoMax 0.1% Gelatin Solution</td><td>EMD Millipore</td><td>ES-006-B</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>G7126-500G</td><td>1.25 M stock conentration in water</td></tr><tr><td>Formaldehyde solution</td><td>Sigma</td><td>F8775-500ML</td><td></td></tr><tr><td>TE (Tris EDTA) pH 8.0 1X</td><td>Quality Biological</td><td>351-011-131</td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride (PMSF) solution</td><td>Sigma</td><td>93482-250ML-F</td><td></td></tr><tr><td>cOmplete Protease Inhibitor Cocktail</td><td>Roche</td><td>11697498001</td><td></td></tr><tr><td>Sodium dodecyl sulfate solution (20%)</td><td>Quality Biological</td><td>A611-0837-10</td><td></td></tr><tr><td>Q125 sonifier</td><td>Qsonica</td><td>4422</td><td></td></tr><tr><td>Triton X-100 solution</td><td>Sigma</td><td>93443-100ML</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma</td><td>30970</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>Dynabeads Protein G for Immunoprecipitation</td><td>Invitrogen</td><td>10004D</td><td></td></tr><tr><td>Dynabeads Protein A for Immunoprecipitation</td><td>Invitrogen</td><td>10002D</td><td></td></tr><tr><td>Dynabeads Protein A/Protein G and Magnet Starter Pack</td><td>Invitrogen</td><td>10015D</td><td></td></tr><tr><td>Lithium chloride</td><td>Sigma</td><td>L4408</td><td></td></tr><tr><td>IGEPAL CA-630</td><td>Sigma</td><td>I8896</td><td></td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Dynabeads mRNA Purification Kit (for mRNA purification from total RNA preps)</td><td>Invitrogen</td><td>61006</td><td></td></tr><tr><td>SuperScript Double-Stranded cDNA Synthesis Kit</td><td>Invitrogen</td><td>11917010</td><td></td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>End-It DNA End-Repair Kit</td><td>Lucigen</td><td>ER81050</td><td></td></tr><tr><td>Klenow Fragment (3&#039;&amp;rarr;5&#039; exo-)</td><td>NEB</td><td>M0212L</td><td></td></tr><tr><td>dATP (10 mM)</td><td>Invitrogen</td><td>18252015</td><td></td></tr><tr><td>T4 DNA ligase</td><td>NEB</td><td>M0202L</td><td></td></tr><tr><td>TrackIt 1 Kb Plus DNA Ladder</td><td>Invitrogen</td><td>10488085</td><td></td></tr><tr><td>E-gel EX agarose gels, 2%</td><td>Invitrogen</td><td>G402002</td><td></td></tr><tr><td>Phusion high-fidelity 2X master mix with HF buffer</td><td>Thermo Fisher</td><td>F531LPM</td><td></td></tr><tr><td>ChIPAb+ Trimethyl-Histone H3 (Lys4) - ChIP Validated Antibody</td><td>EMD Millipore</td><td>17-614</td><td></td></tr><tr><td>Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)</td><td>Abcam</td><td>ab32356</td><td></td></tr><tr><td>Corning Costar Flat Bottom Cell Culture Plates (6-well)</td><td>Fisher</td><td>720083</td><td></td></tr><tr><td>Falcon Standard Tissue Culture Dishes (10cm)</td><td>Fisher</td><td>08772E</td><td></td></tr><tr><td>Bioruptor pico</td><td>Diagenode</td><td>B01010001</td><td></td></tr><tr><td>Qubit Flurometer 2.0</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Thermo Fisher</td><td>Q32851</td><td></td></tr><tr><td>MinElute Reaction Cleanup Kit</td><td>Qiagen</td><td>28204</td><td></td></tr><tr><td>MinElute Gel Extraction Kit</td><td>Qiagen</td><td>28604</td><td></td></tr><tr><td>Anti-Histone H4 (tri methyl K20) antibody - ChIP Grade (ab9053)</td><td>Abcam</td><td>ab9053</td><td></td></tr><tr><td>Labquake rotator</td><td>Thermo Fisher</td><td>400110Q</td><td></td></tr><tr><td>Illumina HiSeq platform</td><td>Illumina</td><td></td><td></td></tr><tr><td>TURBO DNase</td><td>Invitrogen</td><td>AM2238</td><td></td></tr></tbody></table>

carip-seq and chip-seq: methods to identify chromatin-associated rnas and protein-dna interactions in embryonic stem cells

גזע עובריים (ES) תא התחדשות עצמית, בידול נשלטת על ידי אותות חיצוני ורשתות מהותי של גורמי שעתוק epigenetic regulators, שינויים פוסט-תרגום של שינויים היסטוניים combinatorially להשפיע על הגן מצב ביטוי של גנים הסמוכה. RNA הוכח גם לקיים אינטראקציה עם חלבונים שונים כדי לווסת כרומטין דינמיקה של ביטוי גנים. כרומטין-הקשורים RNA immunoprecipitation (CARIP) ואחריו הדור הבא רצפי (CARIP-Seq) היא שיטה סקר RNAs המשויך חלבוני כרומטין, בעוד immunoprecipitation כרומטין ואחריו (הדור הבא רצפי ChIP-Seq) היא טכניקה גנומיקה חזק כדי למפות את המיקום של שינוי post-translational של שינויים היסטוניים, גורמי שעתוק, מכפילי epigenetic בקנה מידה עולמי תאים ES. כאן, אנו מתארים שיטות לבצע CARIP-Seq שבב-Seq, כולל בניית ספריה עבור הדור הבא רצף, ליצירת RNA הקשורים כרומטין הכללית ומפות epigenomic ES תאים.

בהצלחה חקרנו את הכריכה ברמת הגנום של H3K4me3, H3K4me2 ו- KDM5B ES תאים באמצעות פרוטוקול זה צ'יפ-Seq6. ES התאים היו בתרבית בתנאי נטולת מזין (איור 1), קישורים צולבים כמתואר לעיל. Sonication לאחר מכן שבוצעה כמתואר בשלב 3.2 של פרוטוקול שבב-Seq, והוא מוערך על ידי הפעלת הדנ א...

Watch this Scientific Journal Video about CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells at JoVE.com

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

כאן, אנו מתארים שיטות לביצוע שבב-Seq ומפות CARIP-Seq, כולל ספריית לקראת הדור הבא רצפי, ליצירת epigenomic הכללית ו- RNA הקשורים כרומטין תאים ES.

CARIP-Seq ו שבב-Seq: שיטות לזיהוי RNAs הקשורים כרומטין והן אינטראקציות חלבון-DNA בתאי גזע עובריים

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

carip-seq-chip-seq-methods-to-identify-chromatin-associated-rnas

Research

JoVE Journal

Developmental Biology

9.7K Views.  Wayne State University School of Medicine. כאן, אנו מתארים שיטות לביצוע שבב-Seq ומפות CARIP-Seq, כולל ספריית לקראת הדור הבא רצפי, ליצירת epigenomic הכללית ו- RNA הקשורים כרומטין תאים ES.

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

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.

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

The question of how chromatin regulators and chromatin states affect the genome in vivo is key to our understanding of how early cell fate decisions are made in the developing embryo. ChIP-Seq&#8212;the most popular approach to investigate chromatin features at a global level&#8212;is outlined here for Xenopus embryos.

Snapshot הגנום של רגולטורי הכרומטין והברית ב<em&gt; Xenopus</em&gt; עוברים על ידי שבב Seq

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

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

מיפוי רחב של הגנום של אינטראקציות DNA-חלבון עם CHEC-Seq ב<em&gt; Saccharomyces cerevisiae</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Genetics

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

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

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

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

כרומטין פרוטוקול Immunoprecipitation (ChIP) עבור דגימות עובריים נמוך-שפע

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

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

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

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

תקנה אפיגנטיים של לב בידול בתאי גזע עובריים ורקמות

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

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

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

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

הכרומטין immunoprecipitation מן בתאי גזע עובריים אנושיים

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

Biology

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

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

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

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

מיפוי ברזולוציה גבוהה של אינטראקציות חלבון-DNA בתאי גזע עכבר נוירונים באמצעות כרומטין Immunoprecipitation-Exonuclease (ChIP-Exo)

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

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

RNA-Seq ניתוח של ביטוי הגנים בדיפרנציאל חוט השדרה Electroporated אפרוח עוברי

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Neuroscience

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

הכרומטין Assay immunoprecipitation עבור גנים ספציפיים רקמות באמצעות שלבים מוקדמים של עוברי עכברים

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

בידוד הכרומטין על ידי טיהור RNA (ציוץ)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

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

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

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

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

דור של תבנית הכרומטין immunoprecipitation איכות גבוהה עבור ה-DNA רצף תפוקה גבוהה (שבב ואילך)

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

CARIP-Seq ו שבב-Seq: שיטות לזיהוי RNAs הקשורים כרומטין והן אינטראקציות חלבון-DNA בתאי גזע עובריים

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Snapshot הגנום של רגולטורי הכרומטין והברית ב

מיפוי רחב של הגנום של אינטראקציות DNA-חלבון עם CHEC-Seq ב