The work in the Heyer laboratory is supported by grants GM58015 and GM137751 to W.-D.H. Research in the Piazza laboratory is supported by the European Research Council (ERC-StG 3D-loop, grand agreement 851006). D.R. is supported by T32CA108459 and the A.P. Giannini Foundation. We thank Shih-Hsun Hung (Heyer Lab) for sharing his DLC/DLE assay results and for additionally validating the changes to the assays that are detailed in this protocol.

1. Pre-growth, DSB induction, and sample collection
NOTE: Supplementation of all media with 0.01% adenine is recommended for Ade- strains.
<ol>
	<li>Streak out the appropriate haploid strains (see Table 1) on yeast peptone dextrose adenine (YPDA) (1% yeast extract, 2% peptone, 2% glucose, 2% agar, 0.001% adenine) and grow for 2 days at 30 &#176;C.</li>
	<li>Use a single colony to inoculate 5 mL of YPDA in a 15 mL glass culture tube. Grow cultures to saturation at 30 &#176;C with shaking or rotation for aeration.</li>
	<li>DLC assay: Prepare the 5x psoralen stock solution (0.5 mg/mL trioxsalen in 200-proof ethanol) in a fume hood by dissolving psoralen in a 50 mL conical tube wrapped in aluminum foil overnight at room temperature with continuous shaking or inversion. Seal screw top with a transparent film to prevent evaporation. Do not prepare more than 7 mL of 5x psoralen stock solution per 50 mL conical tube to ensure proper dissolution of the psoralen.</li>
	<li>The next day, use 5 mL of the YPDA grown overnight culture to inoculate 50-100 mL of YEP-lactate (1% yeast extract, 2% peptone, 2% w/w lactate, 0.001% adenine) in an appropriately sized flask (budding yeast grows optimally in a flask that is at least 5x the volume of the culture) to an OD600 of ~0.03.</li>
	<li>Grow the culture for ~16 h at 30 &#176;C with shaking at 220 rpm. After ~16 h, measure the OD600 of the culture and it should be ~0.5-0.8. Do not use under- or overgrown cultures.</li>
	<li>For each time point, collect the appropriate volume of cells in a conical tube and place on ice. Typically, this is 1&#160;x 108 cells (approximately 7.5 mL of culture at OD600 1.0 for a haploid wild type strain) for the DLC assay and 5 x 107&#160;cells (approximately 2.5 mL of culture at OD600 1.0) for the DLE assay.</li>
	<li>To ensure the accuracy of the OD600 values, prepare 1:5 dilutions for cultures with an OD600&#8805;0.2 to keep the OD reading at 0.2 or below. For wild type strains, optimal time points for DLC analysis are between 2 h and 6 h, and optimal time points for DLE analysis are between 4 h and 8 h (see Figure 3 and Figure 4).</li>
	<li>DLC assay
	<ol>
		<li>Before each time point, prepare enough 1x psoralen solution (0.1 mg/mL trioxsalen, 50 mM Tris-HCl pH 8.0, 50 mM EDTA pH 8.0, 20% ethanol) in a fume hood for all the samples in a 50 mL conical tube wrapped in foil. Leave at RT.</li>
		<li>Centrifuge the samples at 2,500 x g for 5 min at 4 &#176;C. Resuspend the pellet in 2.5 mL of 1x psoralen solution in a fume hood and transfer to a 60 mm x 15 mm Petri dish. Alternatively, resuspend the pellet in 2.5 mL of TE1 solution (50 mM Tris-HCl pH 8.0, 50 mM EDTA pH 8.0) for a no-crosslinking control.</li>
		<li>Crosslink the samples. For a UV crosslinker fit with long-wave (365 nm) bulbs, position the Petri dishes 1-2 cm below the UV light source with the lid removed atop a plastic or plexiglass plate that has been pre-chilled at &#8722;20&#176; C. For a UV light box, place the Petri dishes directly atop the UV light source. Expose the samples for 10 min with gentle shaking. 
		NOTE: It is recommended to set the UV light source atop an orbital shaker set at ~50 rpm.</li>
		<li>In a fume hood, transfer the sample into a new 15 mL tube. Rinse the Petri dish with 2.5 mL of TE1 solution and add this to the tube. Centrifuge the samples at 2,500 x g for 5 min at 4 &#176;C, properly dispose of the supernatant, and store the pellet at &#8722;20&#176; C. Samples can be stored for up to 1 week before moving to the next step.</li>
	</ol>
	</li>
	<li>DLE assay
	<ol>
		<li>Centrifuge the samples at 2,500 x g for 5 min at 4 &#176;C. Wash the cell pellet in 2.5 mL of cold TE1 solution before repeating the spin and storing the pellets at &#8722;20 &#176;C. Samples can be stored for up to 1 week before moving to the next step.</li>
	</ol>
	</li>
	<li>For sample collection at 0 h, collect the samples prior to the addition of 20% galactose. For subsequent timepoints, induce DSB formation by adding 20% galactose to the cultures to a final concentration of 2%. Collect the remaining samples as described above, pellet, and freeze relative to the time post-DSB induction (i.e., the 4 h sample is collected 4 h after the addition of 20% galactose).</li>
</ol>
2. Cell spheroplasting, lysis, and restriction site restoration
<ol>
	<li>Thaw the samples on ice. Preheat a dry bath to 30 &#176;C.</li>
	<li>Resuspend the samples in 1 mL of spheroplasting buffer (0.4 M sorbitol, 0.4 M KCl, 40 mM sodium phosphate buffer pH 7.2, 0.5 mM MgCl2) and transfer to a 1.5 mL microcentrifuge tube.</li>
	<li>Add 3.5 &#181;L of zymolyase solution (2% glucose, 50 mM Tris-HCl pH 7.5, 5 mg/mL zymolyase 100T; 17.5 &#181;g/mL zymolyase final concentration). Mix gently by tapping or inversion. Incubate at 30 &#176;C for 15 min, and then place on ice. During the 15 min incubation, obtain liquid nitrogen or dry ice.</li>
	<li>Centrifuge for 3 min at 2,500 x g at 4 &#176;C and place the samples on ice. Wash the samples 3x in 1 mL of spheroplasting buffer. Centrifuge the samples for 3 min at 2,500 x g at 4 &#176;C.</li>
	<li>Resuspend the samples in 1 mL of cold 1x restriction enzyme buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 &#181;g/mL BSA pH ~8.0 at RT) and centrifuge for 3 min at 16,000 x g at 4 &#176;C. Place the samples on ice. Repeat the wash 1x.</li>
	<li>Resuspend the samples in 1 mL of cold 1x restriction enzyme buffer. Split the sample (0.5 mL each) into two 1.5 mL microcentrifuge tubes. Centrifuge the samples for 3 min at 16,000 x g at 4 &#176;C.</li>
	<li>Resuspend one tube from each sample in 180 &#181;L of 1.4x restriction enzyme buffer with hybridizing oligos (see Table 2) and one tube in 180 &#181;L of 1.4x restriction enzyme buffer without hybridizing oligos. Each hybridizing oligo is resuspended in 1x TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) and used at a final concentration of 7 nM. The 1x TE replaces the hybridizing oligos in the 1.4x restriction enzyme buffer without hybridizing oligos. 
	NOTE: The hybridizing oligos must be stored at &#8722;20 &#176;C in small aliquots at the working dilution. The concentration of the hybridizing oligos may require optimization; see Discussion.</li>
	<li>Snap freeze the samples in liquid nitrogen or dry ice/ethanol and store at &#8722;80 &#176;C. Samples can be stored at this stage for several months.</li>
</ol>
3. Restriction enzyme digest and intramolecular ligation
<ol>
	<li>Thaw the samples on ice. Preheat one dry bath to 65 &#176;C and another to 37 &#176;C.</li>
	<li>Pipet 36 &#181;L of the sample into a new 1.5 mL microcentrifuge tube on ice. Promptly return the remaining sample to &#8722;80 &#176;C for storage.</li>
	<li>Add 4 &#181;L of 1% SDS (0.1% final concentration) and mix by gently tapping the side of the tube. Incubate at 65 &#176;C for 15 min with gentle tapping every 5 min. Place samples on ice immediately following the incubation. 
	NOTE: This SDS treatment promotes the denaturation of DNA-associated proteins, solubilization of the nuclear envelope, and chromatin accessibility in advance of the restriction enzyme digest and intramolecular ligation steps.</li>
	<li>Add 4.5 &#181;L of 10% Triton X-100 (1% final concentration) and mix by pipetting. Add 20-50 U of restriction enzyme (EcoRI-HF or HindIII-HF) to each sample and incubate at 37 &#176;C for 1 h with gentle agitation every 20-30 min. During this time, preheat a dry bath to 55 &#176;C and preset a water bath to 16 &#176;C.</li>
	<li>Add 8.6 &#181;L of 10% SDS (1.5% final concentration) to each sample and mix by pipetting and tapping. Incubate at 55 &#176;C for 10 min. Add 80 &#181;L of 10% Triton X-100 (6% final concentration) to each sample and mix by pipetting.</li>
	<li>Add 660 &#181;L of 1x ligation buffer without ATP (50 mM Tris-HCl pH 8.0, 10 mM MgCl2, 10 mM DTT, 2.5 &#181;g/mL BSA) + 1 mM ATP pH 8.0 + T4 DNA ligase (8 U/sample) to each sample and mix by gentle inversion. Incubate at 16 &#176;C for 1.5 h with inversion every 30 min. Place the samples on ice immediately following the incubation.</li>
</ol>
4. DNA purification
<ol>
	<li>Preheat one dry bath to 65 &#176;C and another to 37 &#176;C. Add 1 &#181;L of 10 mg/mL proteinase K (prepared in 1x TE pH 8.0) to each sample (12.5 &#181;g/mL final concentration). Incubate at 65 &#176;C for 30 min and place the samples on ice immediately following the incubation until they have cooled.</li>
	<li>Transfer the samples to 2 mL tubes. Working in a fume hood, add an equal volume (~800 &#181;L) of phenol/chloroform/isoamyl alcohol (P/C/IA; pH 8.0) to each sample. Vortex the samples for ~30 s and centrifuge the samples for 5-10 min at 16,000 x g in a microcentrifuge.</li>
	<li>Carefully remove 600 &#181;L of the upper phase of each sample into a new 1.5 mL tube. Properly dispose of the lower phase and 2 mL tubes.</li>
	<li>Precipitate the DNA by adding a 1/10 volume of 3 M sodium acetate pH 5.2 (~60 &#181;L) to each sample, followed by 1 volume of isopropanol (~660 &#181;L). Invert the samples 5x-10x and incubate at RT for 30 min.</li>
	<li>Place the samples on ice for 2 min, and then centrifuge the samples at 16,500 x g for 15 min at 4 &#176;C in a microcentrifuge. Return the samples to ice, pour off the supernatant, and drain the tube on a paper towel.</li>
	<li>Wash the DNA pellet with 200 &#181;L of 70% ethanol. Centrifuge at 16,500 x g for 3 min at 4 &#176;C, place the samples back on ice, pour off the supernatant, and remove the residual alcohol with a pipet. Dry the samples with the caps of the tubes open at 37 &#176;C for 15-20 min.</li>
	<li>Resuspend the DNA pellets in 50 &#181;L of 1x TE by vortexing. Incubate at RT for 30 min, vortex, and then incubate at 37 &#176;C in a dry bath for 30 min. Vortex the samples again, and then place them on ice. Samples can be stored at this stage at &#8722;20 &#176;C for several months, but it is advisable to proceed immediately for the decrosslinking (DLC only) and qPCR steps.</li>
</ol>
5. Psoralen crosslink reversal (for DLC assay only)
<ol>
	<li>Pipet 9 &#181;L of purified DNA into a PCR tube on ice. Add 1 &#181;L of 1 M KOH (0.1 M final concentration). Incubate the samples at 90 &#176;C for 30 min in a thermocycler.</li>
	<li>Add 19.73 &#181;L of sodium acetate solution (0.1 M sodium acetate, 9.6 mM Tris-HCl pH 8.0, 1.0 mM EDTA pH 8.0). Samples can be stored at this stage at &#8722;20 &#176;C for several months, but it is advisable to proceed immediately to the qPCR step.</li>
</ol>
6. Quantitative PCR, controls, and analysis
<ol>
	<li>Using 2 &#181;L of purified DNA, with or without crosslinking, set up a 20 &#181;L qPCR reaction according to the manufacturer&#39;s instructions. Set up each reaction in duplicate. For both the DLC and DLE assays, there are five control reactions and one DLC/DLE quantification reaction, or a total of six reactions per sample, run in duplicate. Supplementary Table S1 and Supplementary Table S2 provide a template for setting up these reactions and analysis, and the sequences of the qPCR primers are listed in Table 3.</li>
	<li>qPCR cycling conditions need to be optimized for each qPCR kit.
	<ol>
		<li>Use the following DLC qPCR conditions, depending on the qPCR kits used: initial denaturation (95 &#176;C for 3 min); 50 rounds of amplification (95 &#176;C for 15 s, 61 &#176;C for 25 s, 72 &#176;C for 15 s with a single acquisition); melting curve analysis (95 &#176;C for 5 s, 65 &#176;C for 1 min, 97 &#176;C with continuous acquisition); and cooling (37 &#176;C for 30 s).</li>
		<li>Use the following qPCR conditions for the DLE assay: initial denaturation (95 &#176;C for 5 min); 50 rounds of amplification (95 &#176;C for 15 s, 60 &#176;C for 30 s, 72 &#176;C for 15 s with a single acquisition); melting curve analysis (95 &#176;C for 5 s, 65 &#176;C for 1 min, 97 &#176;C with continuous acquisition); and cooling (37 &#176;C for 30 s). Note that optimization for different qPCR machines/kits may be required.</li>
	</ol>
	</li>
	<li>DLC assay
	<ol>
		<li>Controls: See the list of qPCR primers in Table 3. A map of the primer binding sites is shown in Figure S1. For supplementary sequence files for the relevant genomic features and amplicons, check the A plasmid Editor (ApE) files; Supplementary Sequence Files 1-5.
		<ol>
			<li>Genomic DNA at ARG4: Use olWDH1760/olWDH1761 to amplify dsDNA located at ARG4. Use this reaction as a loading control&#160;and normalize all other reactions except the DLC signal reaction to this control.</li>
			<li>Intramolecular ligation efficiency at DAP2: Use the 1,904 bp fragment created by EcoRI digestion for intramolecular ligation in parallel with the DLC ligation. Amplification across this ligation junction reports on the intramolecular ligation efficiency and serves as a control to which the DLC signal is normalized.</li>
			<li>DSB induction: Use olWDH1766/olWDH1767 to amplify a region that spans the induced DSB.</li>
			<li>Psoralen crosslinking and resection: Use olWDH2019/olWDH2020 to amplify the unique PhiX region downstream of the EcoRI recognition site. Without crosslink reversal, use the ratio of the ssDNA (no crosslinking) over ARG4 (crosslinked dsDNA) to determine the crosslinking efficiency. With crosslink reversal, resection will lead to a progressive decrease from 1 to 0.5 of the signal relative to ARG4.</li>
			<li>EcoRI recognition site restoration and cutting: Use olWDH1768/olWDH1764 to amplify a region that spans the restored EcoRI recognition site upstream of the DSB on the resected strand. olWDH1769/olWDH1763 amplify a region that spans the EcoRI restriction enzyme site at DAP2. Perform EcoRI cleavage at this site to use as intramolecular ligation control.</li>
		</ol>
		</li>
		<li>DLC signal: Use olWDH1764/olWDH1765 to amplify the chimeric DNA molecule created by intramolecular ligation of the resected (invading) strand and the donor.</li>
		<li>Analysis: Calculate the average and standard deviation of the Cp values for each of the duplicate reactions. Use the ARG4 genomic DNA qPCR Cp values as a reference to normalize all the other control qPCRs. Normalize the DLC signal to the intramolecular ligation control at DAP2. See Figure 3 for typical DLC signal values at 2 h.</li>
	</ol>
	</li>
	<li>DLE assay
	<ol>
		<li>Controls: See the list of qPCR primers in Table 3. A map of the primer binding sites is shown in Figure S1. For supplementary sequence files for the relevant genomic features and amplicons, check the A plasmid Editor (ApE) files (Supplementary Sequence Files 1-5).
		<ol>
			<li>Genomic DNA at ARG4: See section 6.3.1.1.</li>
			<li>Intramolecular ligation efficiency at YLR050C: Use the HindIII digestion to create a 765 bp fragment that will undergo intramolecular ligation in parallel with the DLE ligation. Amplification across this ligation junction reports on the intramolecular ligation efficiency and serves as a control to which the DLE signal is normalized.</li>
			<li>DSB induction: See section 6.3.1.3.</li>
			<li>HindIII recognition site restoration and cutting: Use olWDH2010/olWDH2012 and olWDH2009/2011 to amplify a region that spans the HindIII restriction enzyme sites on the broken strand where it has been resected and extended, respectively.</li>
		</ol>
		</li>
		<li>DLE signal: Use olWDH2009/olWDH2010 to amplify the chimeric DNA molecule created by intramolecular ligation of the resected end of the invading strand upstream of the DSB to the newly extended end downstream of the DSB.</li>
		<li>Analysis: Calculate the average and standard deviation of the Cp values for each of the duplicate reactions. Use the ARG4 genomic DNA qPCR Cp values as a reference to normalize all the other control qPCRs. Normalize the DLE signal to the intramolecular ligation control at YLR050C. Typical DLE signal values at 6 h are reported in Figure 4 and previous publications9.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Symington, L. S., Gautier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-strand+break+end+resection+and+repair+pathway+choice.">Double-strand break end resection and repair pathway choice.</a> Annual Review of Genetics. 45 (1), 247-271 (2011).</li><li>Heyer, W. -. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+recombination+and+genomic+maintenance.">Regulation of recombination and genomic maintenance.</a> Cold Spring Harbor Perspectives in Biology. 7 (8), 016501 (2015).</li><li>Mimitou, E. P., Symington, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sae2,+Exo1+and+Sgs1+collaborate+in+DNA+double-strand+break+processing.">Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing.</a> Nature. 455 (7214), 770-774 (2008).</li><li>Bzymek, M., Thayer, N. H., Oh, S. D., Kleckner, N., Hunter, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+Holliday+junctions+are+intermediates+of+DNA+break+repair.">Double Holliday junctions are intermediates of DNA break repair.</a> Nature. 464 (7290), 937-941 (2010).</li><li>Chen, H., Lisby, M., Symington, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RPA+coordinates+DNA+end+resection+and+prevents+formation+of+DNA+hairpins.">RPA coordinates DNA end resection and prevents formation of DNA hairpins.</a> Molecular Cell. 50 (4), 589-600 (2013).</li><li>Maz&#243;n, G., Symington, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mph1+and+Mus81-Mms4+prevent+aberrant+processing+of+mitotic+recombination+intermediates.">Mph1 and Mus81-Mms4 prevent aberrant processing of mitotic recombination intermediates.</a> Molecular Cell. 52 (1), 63-74 (2013).</li><li>Saini, N., 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=Migrating+bubble+during+break-induced+replication+drives+conservative+DNA+synthesis.">Migrating bubble during break-induced replication drives conservative DNA synthesis.</a> Nature. 502 (7471), 389-392 (2013).</li><li>Piazza, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+processing+of+displacement+loops+during+recombinational+DNA+repair.">Dynamic processing of displacement loops during recombinational DNA repair.</a> Molecular Cell. 73 (6), 1255-1266 (2019).</li><li>Piazza, A., Koszul, R., Heyer, W. -. 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+proximity+ligation-based+method+for+quantitative+measurement+of+D-loop+extension+in+S.+cerevisiae.">A proximity ligation-based method for quantitative measurement of D-loop extension in S. cerevisiae.</a> Methods in Enzymology. 601, 27-44 (2018).</li><li>Sugawara, N., Wang, X., Haber, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+roles+of+Rad52,+Rad54,+and+Rad55+proteins+in+Rad51-mediated+recombination.">In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination.</a> Molecular Cell. 12 (1), 209-219 (2003).</li><li>Renkawitz, J., Lademann, C. A., Kalocsay, M., Jentsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+homology+search+during+DNA+double-strand+break+repair+in+vivo.">Monitoring homology search during DNA double-strand break repair in vivo.</a> Molecular Cell. 50 (2), 261-272 (2013).</li><li>Petukhova, G., Stratton, S., Sung, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalysis+of+homologous+DNA+pairing+by+yeast+Rad51+and+Rad54+proteins.">Catalysis of homologous DNA pairing by yeast Rad51 and Rad54 proteins.</a> Nature. 393 (6680), 91-94 (1998).</li><li>Wright, W. D., Heyer, W. -. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rad54+functions+as+a+heteroduplex+DNA+pump+modulated+by+its+DNA+substrates+and+Rad51+during+D-loop+formation.">Rad54 functions as a heteroduplex DNA pump modulated by its DNA substrates and Rad51 during D-loop formation.</a> Molecular Cell. 53 (3), 420-432 (2014).</li><li>Tavares, E. M., Wright, W. D., Heyer, W. -. D., Cam, E. L., Dupaigne, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+role+of+Rad54+in+Rad51-ssDNA+filament-dependent+homology+search+and+synaptic+complexes+formation.">In vitro role of Rad54 in Rad51-ssDNA filament-dependent homology search and synaptic complexes formation.</a> Nature Communications. 10 (1), 4058 (2019).</li><li>Crickard, J. B., Moevus, C. J., Kwon, Y., Sung, P., Greene, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rad54+drives+ATP+hydrolysis-dependent+DNA+sequence+alignment+during+homologous+recombination.">Rad54 drives ATP hydrolysis-dependent DNA sequence alignment during homologous recombination.</a> Cell. 181 (6), 1380-1394 (2020).</li><li>Lydeard, J. R., Jain, S., Yamaguchi, M., Haber, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Break-induced+replication+and+telomerase-independent+telomere+maintenance+require+Pol32.">Break-induced replication and telomerase-independent telomere maintenance require Pol32.</a> Nature. 448 (7155), 820-823 (2007).</li><li>Jain, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+recombination+execution+checkpoint+regulates+the+choice+of+homologous+recombination+pathway+during+DNA+double-strand+break+repair.">A recombination execution checkpoint regulates the choice of homologous recombination pathway during DNA double-strand break repair.</a> Genes &amp; Development. 23 (3), 291-303 (2009).</li><li>Donnianni, R. A., Symington, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Break-induced+replication+occurs+by+conservative+DNA+synthesis.">Break-induced replication occurs by conservative DNA synthesis.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (33), 13475-13480 (2013).</li><li>Liu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+break-induced+replication+shows+that+it+stalls+at+roadblocks.">Tracking break-induced replication shows that it stalls at roadblocks.</a> Nature. 590 (7847), 655-659 (2021).</li><li>Liu, L., Sugawara, N., Malkova, A., Haber, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+the+kinetics+of+break-induced+replication+(BIR)+by+the+assay+for+monitoring+BIR+elongation+rate+(AMBER).">Determining the kinetics of break-induced replication (BIR) by the assay for monitoring BIR elongation rate (AMBER).</a> Methods in Enzymology. 661, 139-154 (2021).</li><li>Pham, N., 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=Mechanisms+restraining+break-induced+replication+at+two-ended+DNA+double-strand+breaks.">Mechanisms restraining break-induced replication at two-ended DNA double-strand breaks.</a> The EMBO Journal. 40 (10), 104847 (2021).</li><li>Cimino, G. D., Gamper, H. B., Isaacs, S. T., Hearst, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psoralens+as+photoactive+probes+of+nucleic+acid+structure+and+function:+Organic+chemistry,+photochemistry,+and+biochemistry.">Psoralens as photoactive probes of nucleic acid structure and function: Organic chemistry, photochemistry, and biochemistry.</a> Annual Review of Biochemistry. 54 (1), 1151-1193 (1985).</li><li>Yeung, A. T., Dinehart, W. J., Jones, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alkali+reversal+of+psoralen+cross-link+for+the+targeted+delivery+of+psoralen+monoadduct+lesion.">Alkali reversal of psoralen cross-link for the targeted delivery of psoralen monoadduct lesion.</a> Biochemistry. 27 (17), 6332-6338 (1988).</li><li>Shi, Y. B., Spielmann, H. P., Hearst, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Base-catalyzed+reversal+of+a+psoralen-DNA+cross-link.">Base-catalyzed reversal of a psoralen-DNA cross-link.</a> Biochemistry. 27 (14), 5174-5178 (1988).</li><li>Prakash, R., 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=Yeast+Mph1+helicase+dissociates+Rad51-made+D-loops:+Implications+for+crossover+control+in+mitotic+recombination.">Yeast Mph1 helicase dissociates Rad51-made D-loops: Implications for crossover control in mitotic recombination.</a> Genes &amp; Development. 23 (1), 67-79 (2009).</li><li>Liu, 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=Srs2+promotes+synthesis-dependent+strand+annealing+by+disrupting+DNA+polymerase+&#948;-extending+D-loops.">Srs2 promotes synthesis-dependent strand annealing by disrupting DNA polymerase &#948;-extending D-loops.</a> eLife. 6, 22195 (2017).</li><li>Fasching, C. L., Cejka, P., Kowalczykowski, S. C., Heyer, W. -. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top3-Rmi1+dissolve+Rad51-mediated+D+loops+by+a+topoisomerase-based+mechanism.">Top3-Rmi1 dissolve Rad51-mediated D loops by a topoisomerase-based mechanism.</a> Molecular Cell. 57 (4), 595-606 (2015).</li><li>Shah, S. S., Hartono, S. R., Ch&#233;din, F., Heyer, W. -. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisulfite+treatment+and+single-molecule+real-time+sequencing+reveal+D-loop+length,+position,+and+distribution.">Bisulfite treatment and single-molecule real-time sequencing reveal D-loop length, position, and distribution.</a> eLife. 9, 59111 (2020).</li></ol>

The authors have nothing to disclose.

The assays presented allow for the detection of nascent and extended D-loops (DLC assay), D-loop extension (DLE assay), and BIR product formation (DLE assay with no hybridizing oligonucleotides) using proximity ligation and qPCR. ChIP-qPCR of Rad51 to sites distant from the DSB has previously been used as a proxy for Rad51-mediated homology search and D-loop formation. However, this ChIP-qPCR signal is independent of the sequence homology between the break site and a potential donor, as well as the Rad51-associated facto...

Homologous recombination (HR) is a high-fidelity mechanism of repair of DNA double-stranded breaks (DSBs), inter-strand cross-links, and ssDNA gaps, as well as a pathway for DNA damage tolerance. HR differs from error-prone pathways for DNA damage repair/tolerance, such as non-homologous end-joining (NHEJ) and translesion synthesis, in that it utilizes an intact, homologous duplex DNA as a donor to template the repair event. Moreover, many of the key intermediates in the HR pathway are reversible, allowing for exquisite regulation of the individual pathway steps. During the S, G2, and M phases of the cell cycle, HR competes with NHEJ for the repair of the two-ended DSBs1. In addition, HR is essential to DNA replication for the repair of replication-associated DNA damage, including ssDNA gaps and one-sided DSBs, and as a mechanism of DNA lesion bypass2.
A critical intermediate in the HR pathway is the displacement loop, or D-loop (Figure 1). Following end resection, the central recombinase in the reaction, Rad51, loads onto the newly resected ssDNA of the broken molecule, forming a helical filament2. Rad51 then carries out a homology search to identify a suitable homologous donor, typically the sister chromatid in somatic cells. The D-loop is formed when the Rad51-ssDNA filament invades a homologous duplex DNA, which leads to the Watson-Crick base pairing of the broken strand with the complementary strand of the donor, displacing the opposite donor strand. Extension of the 3&#39; end of the broken strand by a DNA polymerase replaces the bases that were lost during the DNA damage event and promotes resolution of the extended D-loop intermediate into a dsDNA product through the synthesis-dependent strand annealing (SDSA), the double-Holliday junction (dHJ), or the break-induced replication (BIR) HR sub-pathways.
Assays that physically monitor the intermediates in the HR pathway permit the analysis of the genetic requirements for each step (i.e., pathway analysis). DSB formation, end resection, dHJs, BIR replication bubbles, and HR products are readily observed by Southern blotting3,4,5,6,7. Yet, Southern blotting fails to report on nascent and extended D-loops, and, thus, an alternative method to reliably measure these joint molecules is required4,8,9. One widely used strategy to analyze nascent D-loop formation is chromatin-immunoprecipitation (ChIP) of Rad51 coupled with quantitative PCR (qPCR)10,11. However, Rad51 association with dsDNA as measured by ChIP-qPCR is independent of sequence homology and the Rad51 accessory factor Rad5410,11. In contrast, an appreciable signal using the method of D-loop analysis presented here, called the D-loop capture (DLC) assay, depends on DSB formation, sequence homology, Rad51, and the Rad51 accessory proteins Rad52 and Rad548. The finding that Saccharomyces cerevisiae Rad51-promoted D-loop formation depends on Rad54 in vivo is&#160;in agreement with numerous in vitro reconstitution experiments indicating that Rad54 is required for homology search and D-loop formation by budding yeast Rad518,12,13,14,15.
Current approaches to measuring D-loop extension, primarily through semi-quantitative PCR, are similarly problematic. A typical PCR-based assay to detect D-loop extension amplifies a unique sequence, resulting from recombination between a break site and an ectopic donor and the subsequent recombination-associated DNA synthesis, via a primer upstream of the region of homology on the broken strand and another primer downstream of the region of homology on the donor strand. Using this method, the detection of recombination-associated DNA synthesis requires the non-essential Pol &#948; processivity factor Pol3216. This finding conflicts with the observation that POL32 deletion has only a mild effect on gene conversion in vivo17. Moreover, these PCR-based assays fail to temporally resolve D-loop extension and BIR product formation, suggesting that the signal results from dsDNA products rather than ssDNA intermediates17,18,19. The D-loop extension (DLE) assay was recently developed to address these discrepancies. The DLE assay quantifies recombination-associated DNA synthesis at a site ~400 base pairs (bp) downstream of the initial 3&#39; invading end9. By this method, D-loop extension is independent of Pol32 and is detectable within 4 h post-DSB induction, whereas BIR products are first observed at 6 h. Indeed, a recent publication from the Haber and Malkova laboratories noted that using this method of preparation of genomic DNA singularly results in ssDNA preservation9,20.
Here, the DLC and DLE assays are described in detail. These assays rely on proximity ligation to detect nascent and extended D-loops in S. cerevisiae (Figure 2)8,9. BIR products can be quantified using this same assay system. For both assays, DSB formation at an HO endonuclease cut site located at the URA3 locus on chromosome (Chr.) V is induced by the expression of the HO endonuclease under the control of a galactose-inducible promoter. Rad51-mediated DNA strand invasion leads to nascent D-loop formation at the site of an ectopic donor located at the LYS2 locus on Chr. II. As the right side of the DSB lacks homology to the donor, repair via SDSA and dHJ formation is not feasible. Initial repair of the DSB by BIR is possible, but the formation of viable products is inhibited by the presence of the centromere21. This deliberate design prevents productive DSB repair, thereby avoiding the resumption of growth by cells with repaired DBSs, which could otherwise overtake the culture during the time course analysis.
In the DLC assay, psoralen crosslinking of the two strands of the heteroduplex DNA within the D-loop preserves the recombination intermediate. Following restriction enzyme site restoration on the broken (resected) strand and digestion, the crosslinking allows for ligation of the unique sequences upstream of the homologous broken and donor DNAs. Using qPCR, the level of chimeric DNA molecule present in each sample is quantified. In the DLE assay, crosslinking is not required, and restriction enzyme site restoration and digestion followed by intramolecular ligation instead link the 5&#39; end of the broken molecule to the newly extended 3&#39; end. Again, qPCR is used to quantify the relative amounts of this chimeric product in each sample. In the absence of restriction enzyme site restoration, the DLE assay reports on the relative levels of the BIR (dsDNA) product that is formed following D-loop extension.
Representative results for each assay using a wild type strain are shown, and readers are referred to Piazza et al.8 and Piazza et al.9 for the use of these assays for the analysis of recombination mutants8,9. The intent of this contribution is to enable other laboratories to adopt the DLC and DLE assays, and support for them is available upon request.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1. Pre-growth, DSB induction, and sample collection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 and 50 mL conical tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL glass culture tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL, 500 mL, or 1 L flasks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm x 15 mm optically clear petri dishes with flat bottom</td>
			<td></td>
			<td></td>
			<td>Suggested: Corning, Catalog Number 430166; or Genesee Scientific, Catalog Number 32-150G</td>
		</tr>
		<tr>
			<td>Benchtop centrifuge with 15 and 50 mL conical tube adapters</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop orbital shaker or tube rotator/revolver</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UV crosslinker or light box with 365 nm UV bulbs set atop an orbital shaker</td>
			<td></td>
			<td></td>
			<td>Suggested: Spectrolinker XL-1500 UV Crosslinker (Spectronics Corporation) or Vilbert Lourmat BLX-365 BIO-LINK, Catalog Number 611110831</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60% w/w sodium DL-lactate syrup</td>
			<td>Sigma-Aldrich</td>
			<td>L1375</td>
			<td>For media preparation</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher</td>
			<td>BP1423500</td>
			<td>For media preparation</td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>BD Difco</td>
			<td>211840</td>
			<td>For media preparation</td>
		</tr>
		<tr>
			<td>Bacto yeast extract</td>
			<td>BD Difco</td>
			<td>212750</td>
			<td>For media preparation</td>
		</tr>
		<tr>
			<td>D-(+)-glucose</td>
			<td>BD Difco</td>
			<td>0155-17-4</td>
			<td>For media preparation</td>
		</tr>
		<tr>
			<td>Trioxsalen</td>
			<td>Sigma-Aldrich</td>
			<td>T6137</td>
			<td>For psoralen cross-linking</td>
		</tr>
		<tr>
			<td>2. Spheroplasting, lysis, and restriction enzyme site restoration</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dry bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen or dry ice/ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated microcentrifuge or microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X restriction enzyme (CutSmart) buffer (500 mM potassium acetate, 200 mM Tris-acetate, 100 mM magnesium acetate, 1 mg/mL BSA, pH 7.8-8.0)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zymolyase 100T</td>
			<td>US Biological</td>
			<td>Z1004</td>
			<td>For spheroplasting</td>
		</tr>
		<tr>
			<td>3. Restriction enzyme digest and intramolecular ligation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI-HF</td>
			<td>New England Biolabs</td>
			<td>R3101</td>
			<td>Restriction enzyme digest for DLC assay</td>
		</tr>
		<tr>
			<td>HindIII-HF</td>
			<td>New England Biolabs</td>
			<td>R3104</td>
			<td>Restriction enzyme digest for DLE assay</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>New England Biolabs</td>
			<td>M0202</td>
			<td>Intramolecular ligation</td>
		</tr>
		<tr>
			<td>4. DNA purification</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 and 2 mL microcentrifuge tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol/chloroform/isoamyl alcohol (P/C/IA) at 25:24:1</td>
			<td>Sigma-Aldrich</td>
			<td>P2069</td>
			<td>DNA purification</td>
		</tr>
		<tr>
			<td>5. Psoralen cross-link reversal</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler/PCR machine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6. qPCR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lightcycler 480</td>
			<td>Roche</td>
			<td>5015278001</td>
			<td>qPCR machine used by the authors</td>
		</tr>
		<tr>
			<td>Lightcycler 96</td>
			<td>Roche</td>
			<td>5815916001</td>
			<td>qPCR machine used by the authors</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 480 96-Well Plate, white</td>
			<td>Roche</td>
			<td>4729692001</td>
			<td>96-well plates for qPCR</td>
		</tr>
		<tr>
			<td>SsoAdvanced Universal SYBR Green Super Mix</td>
			<td>BioRad</td>
			<td>1725271</td>
			<td>qPCR kit used by the authors</td>
		</tr>
		<tr>
			<td>SYBR Green I Master Mix</td>
			<td>Roche</td>
			<td>4707516001</td>
			<td>qPCR kit used by the authors</td>
		</tr>
	</tbody>
</table>

detection of homologous recombination intermediates via proximity ligation and quantitative pcr in saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

DLC assay 
The DLC assay detects both nascent and extended D-loops formed by the invasion of a site-specific DSB into a single donor (Figure 2). Psoralen crosslinking physically links the broken strand and the donor via the heteroduplex DNA within the D-loop. Restriction enzyme site restoration with a long, hybridizing oligo on the resected strand of the break allows for restriction enzyme cleavage, followed by ligation of the broken strand to the proximal dono...

Watch this Scientific Journal Video about Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae at JoVE.com

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by ...

detection-homologous-recombination-intermediates-via-proximity

Research

JoVE Journal

Genetics

1.7K Views.  University of California, Davis. The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

Video: Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (&#916;S) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (&#945;) or a truncated transactivation domain with 7 unique residues (&#946;). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (S&#945;, S&#946;, &#916;S&#945; and &#916;S&#946;) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites.

Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and &#945;/&#946; C-termini, can be quantified.

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 ...

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been investigated in various organs and tissues in rat. However, standard methods for the purification of miRNAs and detection of their expression in rat peritoneal membrane have not been well established. We have developed an effective and reliable method to purify and quantify miRNAs using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in rat peritoneal membrane. This protocol consists of four steps: 1) purification of peritoneal membrane sample; 2) purification of total RNA including miRNA from peritoneal membrane sample; 3) reverse transcription of miRNA to produce cDNA; and 4) qRT-PCR to detect miRNA expression. Using this protocol, we successfully determined that the expression of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) increased significantly in the peritoneal membrane of a rat peritoneal fibrosis model compared with those in control groups. This protocol can be used to study the profile of miRNA expression in the peritoneal membrane of rats in many pathological conditions.

Here we present a protocol for the detection of microRNA expression in rat peritoneal membrane using quantitative real-time reverse-transcription polymerase chain reaction. This method is suitable for studying the microRNA expression profile in rat peritoneal membrane in several pathological conditions.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

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

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

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

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

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

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR

In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). In HIV-1, the antisense protein ASP gene is located on the negative strand of env, in the reading frame -2, spanning the junction gp120/gp41. In the sense orientation, the 3&#39; end of the ASP open reading frame overlaps with gp120 hypervariable regions V4 and V5. The study of ASP RNA has been thwarted by a phenomenon known as RT-self-priming, whereby RNA secondary structures have the ability to prime RT in absence of the specific primer, generating non-specific cDNAs. The combined use of high RNA denaturation with biotinylated reverse primers in the RT reaction, together with affinity purification of the cDNA onto streptavidin-coated magnetic beads, has allowed us to selectively amplify ASP RNA in CD4+ T cells derived from individuals infected with HIV-1. Our method is relatively low-cost, simple to perform, highly reliable, and easily reproducible. In this respect, it represents a powerful tool for the study of antisense transcription not only in HIV-1 but also in other biological systems.

Detection of Human Immunodeficiency Virus Type 1 HIV-1 Antisense Protein ASP RNA Transcripts in Patients by Strand-Specific RT-PCR

RNA hairpins and loops can function as primers for reverse transcription (RT) in absence of sequence-specific primers, interfering with the study of overlapping antisense transcripts. We have developed a technique able to identify strand-specific RNA, and we have used it to study HIV-1 antisense protein ASP.

In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). ...