Name: chromatin immunoprecipitation (chip) using drosophila tissue
Uploaded: 2012-03-23T13:00:00
Description: Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP using Drosophila tissue at JoVE.com

The authors would like to thank Dr. Keji Zhao's lab (NIH/NHLBI) for their help in providing sequencing results. We would also like to thank the UCSC genome project for the use of Genome Browser to visualize mapped sequencing reads. 
This work has been supported by the R00HD055052 NIH Pathway to Independence Award and R01HD065816 from NICHD, the Lucile Parkard Foundation, and the Johns Hopkins University start-up funding to X.C.

(The entire ChIP procedure takes approximately two days. Preparation of ChIP libraries for high-throughput sequencing takes another 2-3 days.)
1. Dissect and Prepare Tissue for ChIP Experiment (~1 million cells)
<ol>
 <li>Dissect tissue of interest (e.g. 200 pairs of Drosophila testes) in cold PBS + 1x protease inhibitor (dissolve 1 pellet of protease inhibitor cocktail in 1.5 mL 1x PBS to obtain 7x stock solution) + PMSF (final concentration 100 &mu;g/mL). Rinse ti...

<ol>
	<li>Kharchenko, P. V., Alekseyenko, A. A., Schwartz, Y. B., Minoda, A., Riddle, N. C., Ernst, J., Sabo, P. J., Larschan, E., Gorchakov, A. A., Gu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+analysis+of+the+chromatin+landscape+in+Drosophila+melanogaster.">Comprehensive analysis of the chromatin landscape in Drosophila melanogaster.</a> Nature. 471, 480-485 (2011).</li><li>Filion, G. J., van Bemmel, J. G., Braunschweig, U., Talhout, W., Kind, J., Ward, L. D., Brugman, W., de Castro, I. J., Kerkhoven, R. M., Bussemaker, H. J., van Steensel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+protein+location+mapping+reveals+five+principal+chromatin+types+in+Drosophila+cells.">Systematic protein location mapping reveals five principal chromatin types in Drosophila cells.</a> Cell. 143, 212-224 (2010).</li><li>Barski, A., Cuddapah, S., Cui, K., Roh, T. Y., Schones, D. E., Wang, Z., Wei, G., Chepelev, I., 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=High-resolution+profiling+of+histone+methylations+in+the+human+genome.">High-resolution profiling of histone methylations in the human genome.</a> Cell. 129, 823-837 (2007).</li><li>Chen, X., Lu, C., Prado, J. R., Eun, S. H., Fuller, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+changes+at+differentiation+gene+promoters+as+they+become+active+in+a+stem+cell+lineage.">Sequential changes at differentiation gene promoters as they become active in a stem cell lineage.</a> Development. 138, 2441-2450 (2011).</li><li>Gonczy, P., Matunis, E., DiNardo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=bag-of-marbles+and+benign+gonial+cell+neoplasm+act+in+the+germline+to+restrict+proliferation+during+Drosophila+spermatogenesis.">bag-of-marbles and benign gonial cell neoplasm act in the germline to restrict proliferation during Drosophila spermatogenesis.</a> Development. 124, 4361-4371 (1997).</li><li>McKearin, D. M., Spradling, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=bag-of-marbles:+a+Drosophila+gene+required+to+initiate+both+male+and+female+gametogenesis.">bag-of-marbles: a Drosophila gene required to initiate both male and female gametogenesis.</a> Genes Dev. 4, 2242-2251 (1990).</li><li>Gan, Q., Schones, D. E., Eun, S. H., Wei, G., Cui, K., Zhao, K., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monovalent+and+unpoised+status+of+most+genes+in+undifferentiated+cell-enriched+Drosophila+testis.">Monovalent and unpoised status of most genes in undifferentiated cell-enriched Drosophila testis.</a> Genome Biol. 11, 42-42 (2010).</li><li>Gan, Q., Chepelev, I., Wei, G., Tarayrah, L., Cui, K., Zhao, K., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+regulation+of+alternative+splicing+and+chromatin+structure+in+Drosophila+gonads+revealed+by+RNA-seq.">Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq.</a> Cell Res. 7, 763-783 (2010).</li></ol>

The versatility of ChIP analyses discussed in this protocol can be used on different tissues, which provides an opportunity to study the chromatin state in a biologically relevant system. ChIP experiments using cells from cultured systems are convenient to perform because large quantity of cells can be easily obtained. However, cultured cells do not necessarily reflect cells in a multi-cellular environment. By developing this technique using dissected tissue from live animals, we can address many questions that cultured ...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Complete Mini protease inhibitor cocktail</td>
 <td>Roche</td>
 <td>11836153001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Formaldehyde (37%)</td>
 <td>Supelco</td>
 <td>47083-U</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PMSF</td>
 <td>Sigma</td>
 <td>78830</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Kontes pellet pestle</td>
 <td>Fischer Scientific</td>
 <td>K749521-1590 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PCR Purification Kit</td>
 <td>Qiagen</td>
 <td>28104</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Linear polyacrylamide</td>
 <td>Sigma</td>
 <td>56575-1ML </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glycogen</td>
 <td>Qiagen</td>
 <td>158930</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>SYBR green/ROX qPCR Master Mix</td>
 <td>Fermentas</td>
 <td>K0223</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mini plate spinner</td>
 <td>Labnet</td>
 <td>Z723533</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Real time PCR system</td>
 <td>Applied Biosystem</td>
 <td>4351101</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Small Volume Ultrasonic Processor</td>
 <td>Misonix</td>
 <td>HS-XL2000</td>
 <td>Model discontinued</td>
 </tr>
 <tr>
 <td>Dynabeads, Protein A</td>
 <td>Invitrogen</td>
 <td>100-01D</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dynamag magnet</td>
 <td>Invitrogen</td>
 <td>123-21D</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phenol:Chlorofrom:IAA</td>
 <td>Invitrogen</td>
 <td>15593-049</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Epicentre DNA END-Repair Kit</td>
 <td>Epicentre Biotechnologies</td>
 <td>ER0720</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MinElute Reaction Cleanup Kit</td>
 <td>Qiagen</td>
 <td>28204</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Klenow Fragment (3'&rarr;5' exo&ndash;)</td>
 <td>New England Biolabs</td>
 <td>M0212S</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>T4 DNA ligase</td>
 <td>Promega Corporation</td>
 <td>M1794</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Adaptor oligonucleotides</td>
 <td>Illumina</td>
 <td>PE-400-1001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Paired-End Primer 1.0 and 2.0</td>
 <td>Illumina</td>
 <td>1001783 
 1001 784</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>E-Gel Electorphoresis system</td>
 <td>Invitrogen</td>
 <td>G6512ST</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>2X Phusion HF Mastermix</td>
 <td>Finnzymes</td>
 <td>F-531</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

chromatin immunoprecipitation (chip) using drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP using Drosophila tissue at JoVE.com

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

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