This work was supported by NIH Grants P50HG004952 and R01GM109099 to MO.

1. Capture Oligonucleotide Design
<ol>
	<li>Design a panel of oligonucleotides to be used during the hybridization capture of the target region/s.
	<ol>
		<li>Aim to design 4&#8211;8 oligonucleotides per target region, but as a minimum, design at least one oligonucleotide targeting each end of the target region.</li>
		<li>If the target sequence is long (&#62;500 base pairs), design the oligonucleotides as spread out as possible to assure effective enrichment of chromatin across the entire target region. 
		NOTE: We have observed optimal captures with 4&#8211;8 oligonucleotides. Even though this process can work well with more oligonucleotides, it is not uncommon to observe a decrease in enrichment yields when too many oligonucleotides are used.</li>
	</ol>
	</li>
	<li>Design oligonucleotides with similar melting temperatures, and make sure that they do not form strong hairpins nor have strong interactions (taking into account that hybridizations will be carried out at 42 &#7506;C). 
	NOTE: If a toehold elution is chosen (4.12.3), an additional external 8 base sequence must be added to the oligonucleotide and will require complementary &#39;release&#39; oligonucleotides at the time of the elution17. Several commercial software tools can be used to select and analyze the oligonucleotides. 25 mers have shown good results, but depending on the genome size and sequence, capture oligonucleotides can contain somewhere between 20&#8211;80 bases. Typically, capture oligonucleotides have a melting temperature close to 62 &#7506;C, but that can change depending on their length. Consistent melting temperatures and/or oligonucleotide length throughout the capture mix can help avoid unwanted biases in enrichment.</li>
	<li>Design the capture oligonucleotides with a biotin moiety (preferably separated by a spacer from the capture sequence) on the 3&#39; end.</li>
</ol>
2. Cell Culture
<ol>
	<li>Grow human lymphoblastoid cells (such as GM12878 shown here) or other cell lines in RPMI 1640 media supplemented with 1% penicillin-streptomycin, 2 mM L-glutamine and 5% fetal bovine serum (FBS), at 37 &#7506;C and 5% CO2.
	<ol>
		<li>Seed initial frozen aliquots (2&#8211;5 x 105 cells) in a T-25 flask with 6 mL of RPMI 1640 media prepared in 2.1.</li>
		<li>Monitor cell density and viability.
		<ol>
			<li>Take a representative aliquot of cells and stain them with an appropriate dye such as trypan blue.</li>
			<li>Measure the cell density using a hemocytometer or an automated cell counter.</li>
		</ol>
		</li>
		<li>Before the cell reaches density of 1 x 106 cells/mL, spin the cells down at 100 x g for 5 min and transfer the cells to a T-75 flask with 25 mL of RPMI 1640 media prepared in 2.1. Grow the cells until cell density approaches 1 x 106 cells/mL.</li>
		<li>Transfer the cells to a T-150 flask in 38 mL of RPMI 1640 media (2.1) and grow the cells for an additional two days. 
		NOTE: Lymphoblastoid cells grow in suspension. General guidelines for growing lymphoblastoid cells recommend cell densities to be kept between 0.2&#8211;1 x 106 cells/mL. Because of the large amount of material required in this technology, the present protocol is purposely written to grow cells to a cell density beyond what is commonly used. Cell growth protocols can be revised and adapted by the reader depending on the cell types to be used.</li>
		<li>Spin the cells down at 100 x g for 5 min, resuspend the cells in 10 mL of RPMI 1640 media (2.1) and pour the suspension into an 850 cm2 roller bottle containing 500 mL of media. Incubate the cells under the same conditions as before but at this point start using constant rotation (~0.5 rpm).</li>
		<li>Monitor the cell growth and change the media when needed (Typically every 3&#8211;4 days). Let the cells grow to a density as close as possible to 2 x 106 cells/mL (1 x 109 cells in total).</li>
		<li>Once the desired cell count is reached, harvest the cells by spinning the cells down at 100 x g for 5 min and resuspend the cells in 36 mL of RPMI 1640 media (2.1). Transfer the cell suspension to a 50 mL conical tube. Take small aliquots for cell counting and DNA extraction.</li>
	</ol>
	</li>
	<li>Crosslink by adding 1 mL of 37 % formaldehyde to reach a final concentration of 1%. 
	CAUTION: Formaldehyde is a carcinogen and should be handled in a fume hood.
	<ol>
		<li>Incubate the cells with rotation (~30&#8211;60 rpm) for 10 min at room temperature (RT).</li>
		<li>Quench the crosslink reaction by adding 2 mL of a 2.5 M glycine solution, for a final concentration of 125 mM.</li>
		<li>Incubate the cells with rotation (~30&#8211;60 rpm) for 5 min at RT. Pellet the cells by centrifuging at 100 x g for 5 min and wash the cells twice with ice cold 1x PBS.</li>
	</ol>
	</li>
	<li>Continue to next step or resuspend the pellet in 5 mL of Cell Lysis Buffer (5 mM Hepes, 85 mM KCl, 0.5% Igepal, protease inhibitors (PI)) and snap-freeze the suspension drop by drop in liquid nitrogen. Store the samples at -80 &#7506;C. 
	NOTE: Prepare &#62;25 mL of Cell Lysis Buffer without Igepal and PI and add those reagents fresh when ready to use. Avoid long-term storage of Cell Lysis Buffer once Igepal and PI have been added. Store Cell Lysis Buffer at 4 &#7506;C for up to 3 months. Igepal is a nonionic, non-denaturing detergent. NP-40 can be potentially used as an alternative to Igepal but we have not used it in this context as NP-40 is not recommended when samples are eventually analyzed by mass spectrometry to characterize the DNA binding proteins.</li>
</ol>
3. Lysis and Shearing
<ol>
	<li>Prepare &#62;15 mL of Nuclei Lysis Buffer (10 mM EDTA, 50 mM Tris pH 8.0, 1 % SDS, PI). 
	NOTE: Add PI fresh, just to the amounts to be used. Avoid long-term storage of Nuclei Lysis Buffer once PI has been added. Nuclei Lysis buffer can be stored at RT for up to 1 month.</li>
	<li>Resuspend the pellet in 20 mL of Cell Lysis Buffer (containing Igepal and PI). Let the pellets thaw on ice and mix well once thawed. Let samples sit for an additional 10 min on ice. Centrifuge the cells at 400 x g at 4 &#7506;C for 5 min.</li>
	<li>Discard the supernatant and resuspend the cells in 6 mL of Nuclei Lysis Buffer with PI (volume can be increased if cells do not form a suspension).</li>
	<li>In preparation for sonication, divide the sample evenly in ~6&#8211;8 microfuge tubes (600 to 1,000 &#181;L for each tube).
	<ol>
		<li>Place the samples on ice or a cold rack and sonicate the sample using a sonicator with a microtip. Use 65% amplitude with 5 x 20 s constant bursts, allowing the suspension to cool down for at least 40 s between pulses. 
		NOTE: If volumes increase &#62;10 mL per sample, a glass cooling cell can be used instead. More and longer bursts would be required then. Sonicators differ greatly. Conditions might have to be optimized by the reader. Other sonication alternatives can be used as well.</li>
	</ol>
	</li>
	<li>Centrifuge the cells at 12,000 x g for 10 min at 4 &#7506;C, transfer the supernatant to a new tube, and discard the pellets.</li>
	<li>Estimate total recovery using a fluorometric method.</li>
	<li>Continue to next step or snap-freeze and store the sample at -80 &#7506;C.</li>
	<li>Optional: Take an aliquot, reverse crosslinks by incubating it overnight at 67 &#7506;C in 0.3 M NaCl. Clean DNA and run it in Bioanalyzer to determine fragment lengths. The bulk of the fragments should be between 500 and 1,000 bp. 
	NOTE: It is OK to stop at this point and store the samples at -80 &#7506;C.</li>
</ol>
4. Hybridization Capture
<ol>
	<li>Prepare 500 mL of 2x Hybridization (Hyb) Buffer (200 mM MES, 2M NaCl, 40 mM EDTA, 0.02 % Tween-20) and 500 mL of Wash Buffer (200 mM NaCl, 0.2 % SDS, 50 mM Tris pH 8). Store the Hyb and Wash Buffers at 4 &#7506;C and room temperature, respectively, for up to 3 months. Use half of the 2x Hyb buffer to prepare a 1x Hybridization Buffer. Also store it at 4 &#7506;C for up to 3 months. 
	NOTE: The total volume of the hybridization capture will be twice the volume of the resulting sample in the previous step (as described so far, the sample volume would be around 6 mL and thus the total hybridization volume would be 12 mL).</li>
	<li>Set aside beads equal to 5 % of the total hybridization capture volume (600 &#181;L in this case). An appropriate and suitable magnet is needed to work with the beads in the following steps.</li>
	<li>Wash the beads three times with 1x Hyb Buffer using twice the beads volume (1200 &#181;L). Resuspend the beads in 1,200 &#181;L of 2x Hyb Buffer. Place the tubes on the magnet until the solution clears and remove the buffer.</li>
	<li>Perform pre-clearing reactions in 1.5 mL microfuge tubes with a maximum of 700 &#181;L of sample per tube. Add enough beads (5%) and 2x Hyb Buffer to dilute the Hyb buffer to 1x (10 tubes with 600 &#181;L of sample, 120 &#181;L of washed beads resuspended in 2x Hyb Buffer, and 480 &#181;L of 2x Hyb Buffer). Incubate them for 10 min at 31 &#7506;C with end over end rotation (~30&#8211;60 rpm).</li>
	<li>Place the tubes on a magnet until beads are immobilized and transfer the samples to new tubes. Discard the beads. 
	NOTE: From this point forward, low-binding tubes are recommended.</li>
	<li>Resuspend the capture oligonucleotides to a working solution of 10 pmol/&#181;L and add 4 &#181;L of this solution to each tube. 
	NOTE: Different regions can act as control for each other, but it is recommended to include a capture oligonucleotide with its sequence scrambled to serve as a true negative control.</li>
	<li>Incubate the tubes for 40 min at 42 &#7506;C with end over end rotation (~30&#8211;60 rpm).</li>
	<li>During the incubation, set aside the beads needed (0.3 &#181;L/pmol of capture oligonucleotide). Wash the beads three times as described in step 4.3, but resuspend the cells in 1 x Hyb Buffer.</li>
	<li>Add the washed beads to the sample and incubate the samples for 30 min at RT with end over end rotation (~30&#8211;60 rpm). Place the tubes on the magnet and remove the supernatant once the beads have been immobilized.</li>
	<li>Washing the beads
	<ol>
		<li>Add 1.2 mL of Wash Buffer and incubate the sample for 5 min with end over end rotation (~30&#8211;60 rpm) at RT. Place tubes on the magnet and wait a couple of min until solution clears. Remove the buffer and repeat this step.</li>
		<li>Add 1.2 mL of Wash Buffer and incubate it for 1 h with end over end rotation (~30-60 rpm) at RT. Place the tubes on the magnet and wait a couple of min until solution clears. Remove the buffer and repeat this step reducing the incubation time to 30 min the second time.</li>
		<li>Add 200 &#181;L of Wash Buffer and incubate it for 15 min with end over end rotation (~30&#8211;60 rpm) at 31 &#7506;C. Place the tubes on the magnet and wait a couple of min until solution clears. Remove the buffer.</li>
		<li>Add 200 &#181;L of PBS and incubate it for 5 min with end over end rotation (~30&#8211;60 rpm) at RT. Place the tubes on the magnet and wait a couple of min until solution clears. Remove the buffer and repeat this step.</li>
	</ol>
	</li>
	<li>Simple elution
	<ol>
		<li>Add 40 &#181;L of molecular grade water to the beads and incubate it at 94 &#7506;C for 5 min.</li>
		<li>Place the tubes on the magnet and wait a couple of minutes until the solution clears.</li>
		<li>Transfer the supernatant into a new tube. Discard the beads.</li>
		<li>Proceed to the proteomic analysis or store the sample at -80 &#7506;C. 
		NOTE: This type of elution works well for later qPCR analysis and some proteomics measurements, but in other instances two alternative approaches described below yield &#34;cleaner&#34; eluates.</li>
	</ol>
	</li>
	<li>DNase elution.
	<ol>
		<li>Add 40 &#181;L of 1x DNase Buffer and 2 U of DNase to each tube.</li>
		<li>Incubate the tubes for 15 min at 37 &#7506;C.</li>
		<li>Place the tubes on a magnet and wait a couple of min until the solution clears.</li>
		<li>Remove the eluted samples from the beads and place the samples in a new low-binding tube.</li>
		<li>Incubate the tube at 94 &#7506;C for 7 min to inactivate the enzyme and proceed to proteomic analyses. 
		NOTE: Lower DNase concentrations can be used if interference with proteomic analyses is observed. It will not be possible to calculate the capture enrichments by qPCR if this elution method is used since the DNase treatment completely destroys the DNA material in the sample.</li>
		<li>Proceed to proteomic analysis or store the sample at -80 &#7506;C.</li>
	</ol>
	</li>
	<li>Toehold elution17. 
	NOTE: This method is not recommended for longer oligonucleotides (&#62;40 bases). This method requires for the capture oligonucleotides to contain an additional 8 bases that do not overlap with the genomic sequence and complementary release oligonucleotides in order to displace the target from the capture oligonucleotides.
	<ol>
		<li>Dilute 2 nanomoles of release oligonucleotides in 40 &#181;L of SSC buffer.</li>
		<li>Incubate it for 20 min at RT.</li>
		<li>Place the tubes on a magnet and wait a couple of minutes until the solution clears.</li>
		<li>Remove the solution from the beads.</li>
		<li>Proceed to proteomic analysis or store the sample at -80 &#7506;C.</li>
	</ol>
	</li>
</ol>
5. Evaluation of Capture Yield by qPCR with Primers for the Target Capture Region of Interest
<ol>
	<li>Use an appropriate software or online tool to design primers and probes for the target region. 
	NOTE: In this experiment, the promoter region of the DUSP3 and DUSP5 were targeted. The primers and probes used are the following. DUSP3: Primer 1 - GTG TTT AGG TTC CCT GAT CC, primer 2 - CGG AAT GTC TGC CTT CG, FAM-labeled probe - TCC ATG CCC GAA TTG TGA CGT CGG; DUSP5: Primer 1 - TGA GAA AGC CCT GAT GTG TC, primer 2 - GCC TTC AGA AAC CCC AAA AG, FAM-labeled probe TGC ATT TAG AGC AGC CAG ATG AGG TT.</li>
	<li>Take an aliquot of the eluate (no more than 10 &#181;L), and dilute 1:10 with molecular grade H2O.</li>
	<li>Extract the DNA of one of the aliquots taken in step 2.1.7 and use that DNA sample to make a standard curve by doing five serial 1:10 dilutions in molecular grade H2O.</li>
	<li>Prepare a qPCR master mix (wide variety of commercial alternatives), add primers and probes and dispense appropriate amount (15 &#181;L if running a 20 &#181;L reaction) in each well.</li>
	<li>Add 5 &#181;L of either: a) diluted sample, b) the standard curve or c) molecular grade H2O (to serve as no template control) to each well.</li>
	<li>Seal the plate and centrifuge it at 100 x g for 1 min.</li>
	<li>Run in a qPCR system with the following parameters: 5 min at 95 &#7506;C followed by 40 cycles of 95 &#7506;C for 20 s, 59 &#7506;C for 20 s and 72 &#7506;C for 25 s.</li>
	<li>Use the standard curve to asses yields and scrambled capture to assess capture specificity.</li>
</ol>

<ol>
	<li>Consortium, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+encyclopedia+of+DNA+elements+in+the+human+genome.">An integrated encyclopedia of DNA elements in the human genome.</a> Nature. 489 (7414), 57-74 (2013).</li><li>Galas, D. J., Schmitz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNAse+footprinting:+a+simple+method+for+the+detection+of+protein-DNA+binding+specificity.">DNAse footprinting: a simple method for the detection of protein-DNA binding specificity.</a> Nucleic Acids Res. 5 (9), 3157-3170 (1978).</li><li>Simon, J. M., Giresi, P. G., Davis, I. J., Lieb, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+formaldehyde-assisted+isolation+of+regulatory+elements+(FAIRE)+to+isolate+active+regulatory+DNA.">Using formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate active regulatory DNA.</a> Nat. Protoc. 7 (2), 256-267 (2012).</li><li>Dai, Y., Kennedy-Darling, J., Shortreed, M. R., Scalf, M., Gasch, A. P., Smith, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Sequence-Specific+Capture+of+Chromatin+and+Mass+Spectrometric+Discovery+of+Associated+Proteins.">Multiplexed Sequence-Specific Capture of Chromatin and Mass Spectrometric Discovery of Associated Proteins.</a> Anal. Chem. 89 (15), 7841-7846 (2017).</li><li>Guillen-Ahlers, H., 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=HyCCAPP+as+a+tool+to+characterize+promoter+DNA-protein+interactions+in+Saccharomyces+cerevisiae.">HyCCAPP as a tool to characterize promoter DNA-protein interactions in Saccharomyces cerevisiae.</a> Genomics. 107 (6), (2016).</li><li>Kennedy-Darling, J., 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=Discovery+of+Chromatin-Associated+Proteins+via+Sequence-Specific+Capture+and+Mass+Spectrometric+Protein+Identification+in+Saccharomyces+cerevisiae.">Discovery of Chromatin-Associated Proteins via Sequence-Specific Capture and Mass Spectrometric Protein Identification in Saccharomyces cerevisiae.</a> J Proteome Res. 13 (8), 3810-3825 (2014).</li><li>Guillen-Ahlers, H., Shortreed, M. R. R., Smith, L. M. M., Olivier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+methods+for+the+analysis+of+chromatin-associated+proteins.">Advanced methods for the analysis of chromatin-associated proteins.</a> Physiol Genomics. 46 (13), 441-447 (2014).</li><li>Lambert, J. -. P., Fillingham, J., Siahbazi, M., Greenblatt, J., Baetz, K., Figeys, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+budding+yeast+chromatin-associated+interactome.">Defining the budding yeast chromatin-associated interactome.</a> Mol Syst Biol. 6, 448 (2010).</li><li>Soldi, M., Bonaldi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Proteomic+Investigation+of+Chromatin+Functional+Domains+Reveals+Novel+Synergisms+among+Distinct+Heterochromatin+Components.">The Proteomic Investigation of Chromatin Functional Domains Reveals Novel Synergisms among Distinct Heterochromatin Components.</a> Mol Cell Proteomics. 12 (3), 764-780 (2013).</li><li>Wang, C. I., 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=Chromatin+proteins+captured+by+ChIP-mass+spectrometry+are+linked+to+dosage+compensation+in+Drosophila.">Chromatin proteins captured by ChIP-mass spectrometry are linked to dosage compensation in Drosophila.</a> Nat Struct Mol Biol. 20 (2), 202-209 (2013).</li><li>Byrum, S. D., Raman, A., Taverna, S. D., Tackett, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChAP-MS:+A+Method+for+Identification+of+Proteins+and+Histone+Posttranslational+Modifications+at+a+Single+Genomic+Locus.">ChAP-MS: A Method for Identification of Proteins and Histone Posttranslational Modifications at a Single Genomic Locus.</a> Cell Rep. 2 (1), 198-205 (2012).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+specific+genomic+regions+and+identification+of+associated+immunoprecipitation+(+eChIP+)+using+CRISPR.">Efficient isolation of specific genomic regions and identification of associated immunoprecipitation ( eChIP ) using CRISPR.</a> Biochem. Bioph. Res. Co. 439 (1), 132-136 (2013).</li><li>Liu, X., 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=In+Situ+Capture+of+Chromatin+Interactions+by+Biotinylated+dCas9.">In Situ Capture of Chromatin Interactions by Biotinylated dCas9.</a> Cell. 170 (5), 1028-1043 (2017).</li><li>Myers, S. A., Wright, J., Zhang, F., Carr, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-APEX-mediated+proximity+labeling+enables+discovery+of+proteins+associated+with+a+predefined+genomic+locus+in+living+cells.">CRISPR/Cas9-APEX-mediated proximity labeling enables discovery of proteins associated with a predefined genomic locus in living cells.</a> bioRxiv. , (2017).</li><li>D&#233;jardin, J., Kingston, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Proteins+Associated+with+Specific+Genomic+Loci.">Purification of Proteins Associated with Specific Genomic Loci.</a> Cell. 136 (1), 175-186 (2009).</li><li>Buxton, K. E., 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=Elucidating+Protein-DNA+Interactions+in+Human+Alphoid+Chromatin+via+Hybridization+Capture+and+Mass+Spectrometry.">Elucidating Protein-DNA Interactions in Human Alphoid Chromatin via Hybridization Capture and Mass Spectrometry.</a> J. Proteome Res. 16 (9), 3433-3442 (2017).</li><li>Kennedy-Darling, J., Holden, M. T., Shortreed, M. R., Smith, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+programmable+release+of+captured+DNA.">Multiplexed programmable release of captured DNA.</a> Chembiochem. 15 (16), 2353-2356 (2014).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+specific+genomic+regions+and+identification+of+associated+molecules+by+engineered+DNA-binding+molecule-mediated+chromatin+immunoprecipitation+(enChIP)+using+CRISPR.">Isolation of specific genomic regions and identification of associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR.</a> Chromatin Protoc. Third Ed. , 43-52 (2015).</li><li>Lambert, J. -. P., Mitchell, L., Rudner, A., Baetz, K., Figeys, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Proteomics+Approach+for+the+Discovery+of+Chromatin-associated+Protein+Networks.">A Novel Proteomics Approach for the Discovery of Chromatin-associated Protein Networks.</a> Mol Cell Proteomics. 8 (4), 870-882 (2009).</li></ol>

There are no conflicts of interest to disclose.

The HyCCAPP method described here has many unique features that make it a powerful approach to uncover DNA-interactions that otherwise would remain elusive. The nature of the process gives HyCCAPP the flexibility to work in different organisms and regions of the genome. It is a method, however, that has several limitations to be considered.
HyCCAPP is a method that avoids any cell modifications so that it can potentially be applied in primary cells, cell culture systems, or even tissue samples...

During the past decade, there has seen a dramatic improvement in sequencing technologies, allowing the study of a wide range of genomes in large numbers of samples, and with astonishing resolution. The Encyclopedia of DNA Elements (ENCODE) Consortium, a large-scale multi-institutional effort spearheaded by the National Human Genome Research Institute of the National Institutes of Health, has provided insights into how individual transcription factors and other regulatory proteins bind to and interact with the genome. The initial effort characterized specific DNA-protein interactions, as assessed by Chromatin immunoprecipitation (ChIP) for over 100 known DNA-binding proteins1. Alternative methods such as DNase footprinting2 and formaldehyde assisted isolation of regulatory elements (FAIRE)3 have also been used to locate specific regions of the genome interacting with proteins, but with the obvious limitation that these experimental approaches do not identify the interacting proteins. Despite the extensive efforts over the past years, no technology has emerged that efficiently allows the comprehensive characterization of protein-DNA interactions in chromatin, and the identification and quantification of chromatin-associated proteins.
To address this need, we developed a novel approach which we termed as Hybridization Capture of Chromatin-Associated Proteins for Proteomics (HyCCAPP). Initially developed in yeast4,5,6, the approach isolates crosslinked chromatin regions of interest (with bound proteins) using sequence-specific hybridization capture. After isolation of the protein-DNA complexes, approaches such as mass spectrometry can be used to characterize the set of proteins bound to the sequence of interest. Thus, HyCCAPP can be considered as a non-biased approach to uncover novel DNA-protein interactions, in the sense that it does not rely on antibodies and it is completely agnostic about the proteins that might be found. There are other approaches capable to uncover novel DNA-interacting proteins7, but most rely on ChIP-like methods8,9,10, plasmid insertions11,12,13,14, or regions with high copy numbers15. In contrast, HyCCAPP can be applied to multi- and single-copy regions, and it does not require any prior information about the proteins in the region. In addition, while some of the methods mentioned above have valuable features, notably avoiding the need for DNA-protein crosslinking reactions, the unique feature of HyCCAPP is that it can be applied to single-copy regions in unmodified cells, and without any prior knowledge about putative binding proteins, or available antibodies.
At this point, HyCCAPP has predominantly been applied to the analysis of various genomic regions in yeast4,5,6, and was recently used to analyze protein-DNA interactions in alpha-satellite DNA, a repeat region in the human genome16. As part of our ongoing work, we have adapted the hybridization capture approach initially developed for yeast chromatin to be applicable to the analysis of human cells, and present here a modified protocol that allows the selective capture of single-copy target regions in the human genome with efficiencies similar to our initial studies in yeast. This new optimized protocol now allows the adaptation and utilization of the technology to interrogate protein-DNA interactions across the human genome, using mass spectrometry or other analytical approaches.
It is important to emphasize that the HyCCAPP method is meant for the analysis of specific target regions and is not yet suitable for genome-wide analyses. The technology is especially useful when dealing with regions for which there is scarce information about interacting proteins, or when a more comprehensive in-depth analysis of interacting proteins at a specific genome locus is desired. HyCCAPP is meant to uncover DNA-binding proteins but not characterize accurately the specific protein binding sites in genomic DNA. In its current implementation, the methodology does not provide information about the DNA binding sequences or motifs for individual proteins. Therefore, it nicely complements existing technologies such as FAIRE, and may allow the identification of novel binding proteins in genomic regions identified by an initial FAIRE analysis.

<table>
	<tbody>
		<tr>
			<td>PrimerQuest tool</td>
			<td>IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OligoAnalyzer 3.1</td>
			<td>IDT</td>
			<td></td>
			<td>Analyze capture oligonucleotids</td>
		</tr>
		<tr>
			<td>PairFold</td>
			<td>UBC</td>
			<td></td>
			<td>Analyze interactions</td>
		</tr>
		<tr>
			<td>RPMI 1640 media</td>
			<td>Thermo Fisher</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>Thermo Fisher</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Fisher</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess automated cell counter</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>850 cm2 roller bottle&#160;</td>
			<td>Greiner Bio-one</td>
			<td>680058</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller system</td>
			<td>Wheaton</td>
			<td>22-288-525</td>
			<td></td>
		</tr>
		<tr>
			<td>37 % formaldehyde&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Igepal CA-630</td>
			<td>Sigma-Aldrich</td>
			<td>I3021</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P4380</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson digital sonifier SFX 150</td>
			<td>Emerson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 3.0 fluorometer</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA BR assay kit</td>
			<td>Thermo Fisher</td>
			<td>Q32850</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioanalyzer 2100</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent DNA 1000 kit</td>
			<td>Agilent</td>
			<td>5067-1504</td>
			<td></td>
		</tr>
		<tr>
			<td>MES sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>M3885</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 5M</td>
			<td>Thermo Fisher</td>
			<td>AM9760G</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-2 magnet</td>
			<td>Thermo Fisher</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-15 magnet</td>
			<td>Thermo Fisher</td>
			<td>12301D</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads M-280 atreptavidin</td>
			<td>Thermo Fisher</td>
			<td>60210</td>
			<td></td>
		</tr>
		<tr>
			<td>Low binding tubes</td>
			<td>Eppendorf</td>
			<td>22431081</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>SciGene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tube shaker and rotator</td>
			<td>Thermo Fisher</td>
			<td>415110Q</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I (RNase-free)</td>
			<td>New England BioLabs</td>
			<td>M0303</td>
			<td></td>
		</tr>
		<tr>
			<td>SSC buffer 20&#215; concentrate</td>
			<td>Sigma-Aldrich</td>
			<td>S6639</td>
			<td></td>
		</tr>
	</tbody>
</table>

adaptation of hybridization capture of chromatin-associated proteins for proteomics to mammalian cells

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

Due to the need for large input amounts of chromatin for HyCCAPP to succeed, cells are grown to relatively high levels of confluency. Trypan blue staining is used to confirm that cell death rates are moderate (&#60;10 %). In single copy experiments, chromatin content prior to hybridization capture needs to be in the femtomolar range, which usually requires at least 109 cells as starting material. Prior to full scale experiments, it is recommended to test capture oligonucleotide...

Watch this Scientific Journal Video about Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells at JoVE.com

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

adaptation-hybridization-capture-chromatin-associated-proteins-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

6.2K Views.  Texas Biomedical Research Institute. This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

Video: Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

This work describes a protocol for the generation of high resolution in situ Hi-C libraries from tightly staged pre-gastrulation Drosophila melanogaster embryos.

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in mammalian cell lines. Rather than globally assaying removal of xenobiotic-induced or spontaneous chromosomal DPC removal, this assay examines the repair of a homogeneous, chemically defined lesion specifically introduced at one site within a plasmid DNA substrate. Importantly, this approach avoids the use of radioactive materials and is not dependent on expensive or highly-specialized technology. Instead, it relies on standard recombinant DNA procedures and widely available real-time, quantitative polymerase chain reaction (qPCR) instrumentation. Given the inherent flexibility of the strategy utilized, the size of the crosslinked protein, as well as the nature of the chemical linkage and the precise DNA sequence context of the attachment site can be varied to address the respective contributions of these parameters to the overall efficiency of DPC repair. Using this method, plasmids containing a site-specific DPC were transfected into cells and low molecular weight DNA recovered at various times post-transfection. Recovered DNA is then subjected to strand-specific primer extension (SSPE) using a primer complementary to the damaged strand of the plasmid. Since the DPC lesion blocks Taq DNA polymerase, the ratio of repaired to un-repaired DNA can be quantitatively assessed using qPCR. Cycle threshold (CT) values are used to calculate percent repair at various time points in the respective cell lines. This SSPE-qPCR method can also be used to quantitatively assess the repair kinetics of any DNA adduct that blocks Taq polymerase.

The goal of this protocol is to quantify the repair of defined DNA-protein crosslinks on plasmid DNA. Lesioned plasmids are transfected into recipient mammalian cell lines and low-molecular weight harvested at multiple time points post-transfection. DNA repair kinetics are quantified using strand-specific primer extension followed by qPCR.

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in ...

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly being extended to integrative studies to enhance the understanding of molecular dynamics within cells. Current tools for the visualization and integration of proteomics with other omics datasets are inadequate for large-scale studies. Furthermore, they only capture basic sequence identify, discarding post-translational modifications and quantitation. To address these issues, we developed PoGo to map peptides with associated post-translational modifications and quantification to reference genome annotation. In addition, the tool was developed to enable the mapping of peptides identified from customized sequence databases incorporating single amino acid variants. While PoGo is a command line tool, the graphical interface PoGoGUI enables non-bioinformatics researchers to easily map peptides to 25 species supported by Ensembl genome annotation. The generated output borrows file formats from the genomics field and, therefore, visualization is supported in most genome browsers. For large-scale studies, PoGo is supported by TrackHubGenerator to create web-accessible repositories of data mapped to genomes that also enable an easy sharing of proteogenomics data. With little effort, this tool can map millions of peptides to reference genomes within only a few minutes, outperforming other available sequence-identity based tools. This protocol demonstrates the best approaches for proteogenomics mapping through PoGo with publicly available datasets of quantitative and phosphoproteomics, as well as large-scale studies.

Here we present the proteogenomic tool PoGo and protocols for fast, quantitative, post-translational modification and variant enabled mapping of peptides identified through mass spectrometry onto reference genomes. This tool is of use to integrate and visualize proteogenomic and personal proteomic studies interfacing with orthogonal genomics data.

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly ...

CRISPR Guide RNA Cloning for Mammalian Systems

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative strategies for guide RNA (gRNA) cloning are outlined based on the usage of the Type IIS restriction enzyme BsmBI in combination with a set of compatible vectors. Outside of the access to Sanger sequencing services to validate the generated vectors, no special equipment or reagents are required aside from those that are standard to modern molecular biology laboratories. The outlined method is primarily intended for cloning one single gRNA or one paired gRNA-expressing vector at a time. This procedure does not scale well for the generation of libraries containing thousands of gRNAs. For those purposes, alternative sources of oligonucleotide synthesis such as oligo-chip synthesis are recommended. Finally, while this protocol focuses on a set of mammalian vectors, the general strategy is plastic and is applicable to any organism if the appropriate gRNA vector is available.

Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined.

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...