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 tissue twice and resuspend tissue in 200 &mu;L of the same PBS solution with inhibitors.</li>
 <li>To fix the sample, add 5.5 &mu;L 37% formaldehyde (warm formaldehyde in 37 &deg;C water bath for 1 min before using). Incubate at 37 &deg;C for 15 minutes, vortex every 5 minutes in between.</li>
 <li>Place the sample on ice for the tissue to settle for 2 minutes. Rinse 2x with 450 &mu;L PBS (with protease inhibitors and PMSF). Sample can now be stored at -20 &deg;C. Repeat dissection several times to obtain enough material for ChIP.</li>
</ol>
2. Prepare Supernatant with Protein-DNA Conjugation for ChIP Assay
<ol>
 <li>Combine enough sample from step 1.3 in a 1.75 mL eppendorf tube.</li>
 <li>Remove PBS from tissue sample. Add 200 &mu;L of lysis buffer (50 mM Tris-HCl, pH 7.6, 1 mM CaCl2, 0.2% Triton X-100 or NP-40, 5 mM butyrate, and 1x proteinase inhibitor cocktail). Add fresh PMSF stock solution to lysis buffer (PMSF final concentration of 0.5 mM). Homogenize with blue homogenizer (Fisher Cat# 749521-1590) until tissues dissociate completely without any aggregation, incubate at RT (Room Temperature) for 10 minutes.</li>
 <li>Sonication: use microtip sonicator (Misonix HS-XL200) at power 20. Sonicate for 10 sec and rest on ice for 50 sec. Repeat 4-5 times (optimal sonication time should be determined empirically, as longer sonication will yield smaller fragments. For ChIP using antibodies against histone modifications, we normally sonicate chromatin to approximately 200-300 bp; for transcription factor or chromatin regulator (e.g. Polycomb proteins), we normally sonicate chromatin to 200-1000 bp. However, the optimal fragment size should be tested empirically. Note: ALWAYS KEEP THE TUBE ON ICE EVEN DURING SONICATING TO PREVENT SAMPLE FROM HEATING UP!</li>
 <li>Dilute sample by adding 1.8 mL RIPA buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton X-100, with protease inhibitors and PMSF at the same concentration as described previously). Save 40 &mu;L as input control by adding 2 &mu;L 5M NaCl and incubate at 65 &deg;C overnight (O/N) to reverse crosslink.</li>
 <li>To conjugate antibody to beads, add 40 &mu;L Protein A beads to a 1.5 ml eppendorf tube, then add 600 &mu;L PBS and rock at 4 &deg;C for 2 minutes, apply beads to magnet and remove supernatant. Add 100 &mu;L of PBS and the antibody of interest (amount of antibody should be determined empirically). Incubate at RT for 1 hour or 4 &deg;C for 4 hours.</li>
 <li>Remove supernatant from beads by magnet and add 1mL chromatin extract from step 2.4 to the beads. Rotate at 4 &deg;C O/N.</li>
 <li>Apply ChIP sample to magnet and remove supernatant. Wash the beads with the following buffer at 4 &deg;C for 10 minutes each: 
 2x with 1 mL of RIPA buffer [1.89 mL 'RIPA buffer' + 315 &mu;L 7x protease inhibitors + 20 &mu;L PMSF]; 
 2x with 1 mL of RIPA buffer + 0.3 M NaCl [1.89 mL RIPA buffer + 220 &mu;L 3 M NaCl]; 
 2x with 1 mL of LiCl buffer (0.25 M LiCl, 0.5% NP40, 0.5% NaDOC); 
 1x with 1 mL of 1x TE + 0.2% Triton X-100; 
 1x with 1 mL of 1x TE. </li>
 <li>To reverse crosslink, resuspend the beads in 100 &mu;L TE buffer + 3 &mu;L 10% SDS + 5 &mu;L of proteinase K (20 mg/mL). Incubate at 65 &deg;C O/N.</li>
 <li>Apply beads to magnet and transfer supernatant into a new tube. THE SUPERNATANT CONTAINS YOUR DNA SAMPLE. Wash beads with 100 &mu;L TE + 0.5 M NaCl and combine the two supernatant.</li>
 <li>Phenol-chloroform extract the DNA sample. Add 200 &mu;L Phenol:Chlorofrom:IAA (25:24:1) mix and vortex. Spin at 14k for 5 minutes at RT. Alternatively, DNA can be extracted using Qiagen PCR purification kit.</li>
 <li>Transfer the supernatant/aqueous layer into a new tube, and add linear acrylamide to a final concentration of 20 &mu;g/mL (Alternatively 1 &mu;L of glycogen at 20 mg/mL for every 1 mL of supernatant can also be used. Glycogen is used for subsequent qPCR or PCR analyses while linear acrylamide is used for sequencing assays.) Add 20 &mu;L of 3M NaOAc and 500 &mu;L 100% EtOH. Mix well and incubate at 80 &deg;C for 10 minutes. Spin at maximum speed for 20 minutes at 4 &deg;C.</li>
 <li>Remove supernatant and wash with 300 &mu;L 70% EtOH. Air dry and resuspend sample in 50 &mu;L TE buffer. Sample can now be stored at -20 &deg;C. Sample can be used for quantitative PCR (qPCR) assay (Figure 1).</li>
</ol>
3. Analyze ChIP-ed DNA
3a. Analyze ChIP-ed DNA using qPCR
<ol>
 <li>Dilute genomic DNA at the following concentration to make a standard curve: undiluted, 1/10, 1/100, 1/1000, 1/5000.</li>
 <li>For each primer pair, set up qPCR in duplicate in a 96-well plate with genomic DNA from step 3a.1, input control and ChIP-ed DNA: 
 10 &mu;L 2x SYBR PCR mix (Fermentas, K0222); 
 1 &mu;L 10 &mu;M forward primer; 
 1 &mu;L 10 &mu;M reverse primer; 
 x &mu;L ChIP-ed DNA (input control, or genomic DNA); 
 y &mu;L nuclease-free H2O to adjust the reaction volume to 20 &mu;L. </li>
 <li>Spin down the 96-well plate with Mini plate spinner (Labnet).</li>
 <li>Perform qPCR with ABI 7300 real time PCR system (or other real time PCR systems) using the following condition: 
 Stage 1: 50 &deg;C for 2 min, 1 cycle; 
 Stage 2: 95 &deg;C for 10 min, 1 cycle; 
 Stage 3: 95 &deg;C for 15 s, 60 &deg;C for 1 min, 40 cycles; 
 Stage 4 (dissociation stage): 95 &deg;C for 15 s, 60 &deg;C for 1 min, 95 &deg;C for 15 s. </li>
 <li>Check the dissociation curve: One peak at the thermal dissociation plot suggests a single amplicon from the PCR reaction. More than one peak indicates non-specific product with the primer pair, the qPCR data should not be used.</li>
 <li>Determine the linear phase of exponential amplification of PCR reaction for each primer set, by calculating the formula that resolves DNA amount according to the Ct (cycle threshold) value, which is based on the standard curve using the series of diluted genomic DNA in 3a.1.</li>
 <li>If the Ct value of ChIP-ed DNA and input control are within the linear range of Ct value, calculate the enrichment of ChIP-ed DNA versus input, according to the diluting factor of ChIP-ed DNA and input control in 2.4.</li>
</ol>
3b. Amplify ChIP-ed DNA for high throughput sequencing.
<ol>
 <li>End-repair of ChIP-ed DNA mix (Use Epicentre DNA END-Repair kit), set up on ice: 
 1-34 &mu;l genomic DNA from step 2.12 (0.1 to 5 &mu;g) 
 5 &mu;l 10x End repair buffer 
 5 &mu;l 2.5 mM dNTP mix 
 5 &mu;l 10 mM ATP 
 x &mu;l H2O to adjust the reaction volume to 49 &mu;l 
 1 &mu;l End-Repair Enzyme mix (T4 DNA Polymerase + T4 Polynucleotide Kinase) 
 Incubate for 45 minutes at RT. Purify reaction using Qiagen MinElute Reaction 
 Cleanup kit. Elute in 30 &mu;l Elution Buffer (EB). </li>
 <li>Add A-overhangs to the 3' end: 
 30 &mu;l of eluted DNA product from step 3b.1 
 5 &mu;l 10x NEB Buffer #2 
 10 &mu;l 1 mM dATP 
 2 &mu;l dH2O 
 3 &mu;l 5 U/ &mu;l Klenow Fragment (3'&rarr;5' exo-) 
 Incubate for 30 minutes at 37 &deg;C. Purify reaction using MinElute ReactionCleanup kit. Elute in 10 &mu;l EB. </li>
 <li>Solexa linker ligation: 
 10 &mu;l of eluted DNA from step 3b.2 
 10 &mu;l ddH2O 
 2.5 &mu;l 10x T4 DNA ligase buffer 
 1 &mu;l Adaptor oligo mix from Illumina (1/10-diluted from stock) 
 2.5 &mu;l T4 DNA ligase (400 units/&mu;l) 
 Incubate for 1 hour at RT. Purify reaction using MinElute ReactionCleanup kit. Elute in 20 &mu;l EB. Sample can now be stored at -20 &deg;C.</li>
 <li>Run the eluted DNA sample from step 3b.3 using the E-gel apparatus. Isolate the 300-500 bp band in the gel and purify using the Qiagen Gel Extraction kit (this step eliminates the ~125 bp self-ligated linkers from the adaptor oligo ligation). Elute in 12 &mu;l EB. </li>
 <li>Amplify library using paired-end (PE) primers from Illumina: 
 10.5 &mu;l of eluted DNA from step 3b.4 
 12.5 &mu;l of master mix (2X Phusion HF, Finnzymes) 
 1 &mu;l of PE1 PCR primer 1 
 1 &mu;l of PE2 PCR primer 2 
 PCR condition: 
 98 &deg;C for 30 sec 
 (98 &deg;C 10 sec, 65 &deg;C 30 sec, 72 &deg;C 30 sec), repeat 20 cycles 
 72&deg;C for 5 minutes. </li>
 <li>Samples from step 3b.5 can be used for ChIP-seq after selecting the appropriate sized DNA (300-500 bp) using the standard 2% agarose gel. Sample can now be stored at -20 &deg;C. It is best to isolate each ChIP sample on a single gel to prevent contamination. Samples can be submitted for high-throughput sequencing.</li>
</ol>
3c. Solexa pipeline analysis
<ol>
 <li>The 25-bp sequencing reads were obtained from the Illumina Genome Analyzer (GA) pipeline.</li>
 <li>All reads were aligned to the Drosophila genome (dm3) using the ELAND (Efficient Local Alignment of Nucleotide Data) software, allowing up to two mismatches with the reference sequence.</li>
 <li>Uniquely mapped reads were retained for downstream analysis. For multiple identical reads, up to three copies were retained to reduce the possibility of biases from PCR amplification.</li>
 <li>The output of the GA Pipeline was converted to browser extensible data (BED) files.</li>
 <li>To generate the wig files for uploading to the UCSC browser for visualization, we used a python scrip which has been described in a previous publication3 with 4 bp as the window size and 160 bp as the DNA fragment size.</li>
 <li>To classify Drosophila genes into silent and expressed genes, we used a digital number called RPKM (reads/per kilobase merged exonic region/per million mapped reads). Genes with RPKM = 0 were classified as silent group and genes with RPKM &ge; 1 were classified into the expressed groups, which can be classified into three subgroups as genes with low, moderate and high expression levels, and each group has about the same number of genes.</li>
 <li>To compare the histone modification level and gene expression, we used a python script which has been described in a previous publication3.</li>
</ol>
4. Representative Results
Examples of ChIP-qPCR results using bam (bag of marbles) mutant testes are shown in Figure 14. In bam testes, there is a failure in the transition from proliferative spermatogonia to differentiating spermatocytes5, 6. We use bam testes as a source for undifferentiated germ cells, which are enriched in this tissue type. Differentiation genes required for sperm differentiation, such as male-specific transcription factor 87 (mst87F), don juan (dj), and fuzzy onion (fzo) are not expressed in bam testes. These genes are highly enriched with the repressive H3K27me3 histone modification7 (Figure 1A), but are devoid of the active H3K4me3 histone modification8 (Figure 1B), a chromatin signature we called 'monovalent'7. Enrichment of either H3K27me3 or H3K4me3 is determined by normalization to a constitutively expressed Cyclin A (CycA) gene4.
ChIP-seq analysis using the same set of antibodies (i.e. repressive H3K27me3 and active H3K4me3) with bam mutant testes (Figure 2) validated the qPCR results shown in Figure 1. For the three tested terminal differentiation genes mst87F, dj and fzo, their genomic regions are highly enriched with H3K27me3 but not H3K4me3 (Figure 2A-2C). In contrast, the constitutively expressed CycA gene has significant H3K4me3 but little H3K27me3 binding near its transcription start site (TSS) (Figure 2D).
Furthermore, the ChIP profiles of H3K27me3 and H3K4me3 near the TSSs of four classes of differentially expressed genes are consistent with the function of each histone modification. As shown in Figure 3A, enrichment of H3K27me3 downstream of the TSSs is inversely correlated with gene expression level. The silent genes have the highest H3K27me3 whereas the highly expressed genes have no H3K27me3 binding. These data is consistent with the repressive role of H3K27me3 on gene expression. In contrast, enrichment of H3K4me3 around the TSSs showed the opposite correlation with gene expression level (Figure 3B), consistent with the active role of H3K4me3 on gene expression.
<img alt="Figure 1" src="/files/ftp_upload/3745/3745fig1.jpg" /> 
 Figure 1. qPCR analysis of ChIP-ed DNA using antibody against either the repressive H3K27me3 histone modification (A) or active H3K4me3 histone modification (B) in undifferentiated cell-enriched bam mutant testes. (A) In bam testes, differentiation genes (Mst87F, dj, fzo) are enriched with the H3K27me3 repressive histone modification. (B) Differentiation genes are depleted with H3K4me3 active mark. The level of ChIP DNA (ChIP DNA/input) at the target gene (Mst87F, dj or fzo) was first normalized to the level of ChIP DNA at the control CycA gene. Error bars indicate standard deviation from three independent biological replicates.
<img alt="Figure 2" src="/files/ftp_upload/3745/3745fig2.jpg" /> 
Figure 2. UCSC genome browser snapshots of H3K27me3 and H3K4me3 enrichment across the entire genomic regions of (A) Mst87F (B) dj and (C) fzo, (D) CycA genes. The circled H3K27me3-enriched region in (D) reflects the chromatin status of an overlapping gene CG7264, which is lowly expressed in bam testes (RPKM=1) but highly expressed in wild-type testes (RPKM=130)8 (ChIP-seq data from7). Probes used in quantitative PCR analysis of ChIP results in Figure 1 are labeled at the bottom of each plot. <a href="https://www.jove.com/files/ftp_upload/3745/3745fig2large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 3" src="/files/ftp_upload/3745/3745fig3.jpg" /> 
Figure 3. ChIP-seq profiles using H3K27me3 and H3K4me3 in bam testes7. The four gene groups (7,509 genes) were classified according to their gene expression levels based on RNA-seq results8. The representative classes of genes are plotted for enrichment of a particular histone modification, using sequences from 3kb upstream to 3kb downstream of their transcription start sites (TSSs). This generates a profile of enrichment of (A) H3K27me3 (K27) and (B) H3K4me3 (K4) ChIP-seq analyses in each group. <a href="https://www.jove.com/files/ftp_upload/3745/3745fig3large.jpg" target="_blank">Click here to view larger figure</a>.

<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>

We have nothing to disclose.

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 ...

chromatin-immunoprecipitation-chip-using-drosophila-tissue

Research

JoVE Journal

Biology

25.0K Views.  Johns Hopkins University. Recentemente high-throughput tecnologia di sequenziamento ha notevolmente aumentato la sensibilità di immunoprecipitazione della cromatina (ChIP) esperimento e ha spinto la sua applicazione con cellule purificate o tessuti sezionati. Qui delineare un metodo per usare la tecnica ChIP con Drosophila Tessuto, che può definire la condizione cromatina endogeno in un sistema ben caratterizzato biologico.

Video: Chromatin Immunoprecipitation ChIP using Drosophila tissue

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

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.

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

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

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.

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

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

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.

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

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...