Name: genome-wide determination of mammalian replication timing by dna content measurement
Uploaded: 2017-01-19T14:00:00
Description: Watch this Scientific Journal Video about Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement at JoVE.com

Wij danken Oriya Vardi voor hulp bij het genereren van cijfers. Werk in de IS-groep werd gesteund door de Israel Science Foundation (subsidie ​​No. 567/10) en de European Research Council Starting Grant (# 281306).

Opmerking: ToR kan alleen worden gemeten op de groei, niet-gesynchroniseerde cellen. De procedure begint met ten minste 1-2 x 10 6 snelgroeiende cellen, die gewoonlijk leiden tot ~ 1 x 10 5 cellen in de S fase (de snelheidsbeperkende stap). Het verdient aanbeveling om elk experiment met twee of drie herhalingen voeren. Het hele proces van CNR-ToR kan worden voltooid binnen een week - twee dagen worden gewijd aan alle trappen naar bibliotheek bereiding 01:59 dagen nodig voor sequentiebepaling en een...

<ol>
	<li>Farkash-Amar, S., Simon, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+the+replication+program+in+mammals.">Genome-wide analysis of the replication program in mammals.</a> Chromosome Res. 18 (1), 115-125 (2010).</li><li>Yaffe, 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=Comparative+analysis+of+DNA+replication+timing+reveals+conserved+large-scale+chromosomal+architecture.">Comparative analysis of DNA replication timing reveals conserved large-scale chromosomal architecture.</a> PLoS Genet. 6 (7), e1001011 (2010).</li><li>Hiratani, 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=Global+reorganization+of+replication+domains+during+embryonic+stem+cell+differentiation.">Global reorganization of replication domains during embryonic stem cell differentiation.</a> PLoS Biol. 6 (10), (2008).</li><li>Rivera-Mulia, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+changes+in+replication+timing+and+gene+expression+during+lineage+specification+of+human+pluripotent+stem+cells.">Dynamic changes in replication timing and gene expression during lineage specification of human pluripotent stem cells.</a> Genome Res. 25 (8), 1091-1103 (2015).</li><li>Ryba, T., 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=Abnormal+developmental+control+of+replication-timing+domains+in+pediatric+acute+lymphoblastic+leukemia.">Abnormal developmental control of replication-timing domains in pediatric acute lymphoblastic leukemia.</a> Genome Res. 22 (10), 1833-1844 (2012).</li><li>Farkash-Amar, 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=Global+organization+of+replication+time+zones+of+the+mouse+genome.">Global organization of replication time zones of the mouse genome.</a> Genome Res. 18 (10), 1562-1570 (2008).</li><li>Koren, A., McCarroll, 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=Random+replication+of+the+inactive+X+chromosome.">Random replication of the inactive X chromosome.</a> Genome Res. 24 (1), 64-69 (2014).</li><li>Mukhopadhyay, 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=Allele-specific+genome-wide+profiling+in+human+primary+erythroblasts+reveal+replication+program+organization.">Allele-specific genome-wide profiling in human primary erythroblasts reveal replication program organization.</a> PLoS Genet. 10 (5), e1004319 (2014).</li><li>McNairn, A. J., Gilbert, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenomic+replication:+linking+epigenetics+to+DNA+replication.">Epigenomic replication: linking epigenetics to DNA replication.</a> Bioessays. 25 (7), 647-656 (2003).</li><li>Sima, J., Gilbert, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+correlations:+replication+timing+and+mutational+landscapes+during+cancer+and+genome+evolution.">Complex correlations: replication timing and mutational landscapes during cancer and genome evolution.</a> Curr Opin Genet Dev. 25, 93-100 (2014).</li><li>Kenigsberg, 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=The+mutation+spectrum+in+genomic+late+replication+domains+shapes+mammalian+GC+content.">The mutation spectrum in genomic late replication domains shapes mammalian GC content.</a> Nucleic Acids Res. 44 (9), 4222-4232 (2016).</li><li>Woo, Y. H., Li, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+replication+timing+and+selection+shape+the+landscape+of+nucleotide+variation+in+cancer+genomes.">DNA replication timing and selection shape the landscape of nucleotide variation in cancer genomes.</a> Nat Commun. 3, 1004 (2012).</li><li>Liu, L., De, S., Michor, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+replication+timing+and+higher-order+nuclear+organization+determine+single-nucleotide+substitution+patterns+in+cancer+genomes.">DNA replication timing and higher-order nuclear organization determine single-nucleotide substitution patterns in cancer genomes.</a> Nat Commun. 4, 1502 (2013).</li><li>Goren, A., Cedar, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicating+by+the+clock.">Replicating by the clock.</a> Nat Rev Mol Cell Biol. 4 (1), 25-32 (2003).</li><li>Selig, S., Okumura, K., Ward, D. C., Cedar, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delineation+of+DNA+replication+time+zones+by+fluorescence+in+situ+hybridization.">Delineation of DNA replication time zones by fluorescence in situ hybridization.</a> EMBO J. 11 (3), 1217-1225 (1992).</li><li>Smith, L., Thayer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+replicating+timing+combined+with+fluorescent+in+situ+hybridization.">Chromosome replicating timing combined with fluorescent in situ hybridization.</a> J Vis Exp. (70), e4400 (2012).</li><li>Simon, 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=Asynchronous+replication+of+imprinted+genes+is+established+in+the+gametes+and+maintained+during+development.">Asynchronous replication of imprinted genes is established in the gametes and maintained during development.</a> Nature. 401 (6756), 929-932 (1999).</li><li>Phi-Wilson, J. T., Recktenwald, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coating+agents+for+cell+recovery.">Coating agents for cell recovery.</a> Google Patents. , (1993).</li><li>Koren, 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=Differential+relationship+of+DNA+replication+timing+to+different+forms+of+human+mutation+and+variation.">Differential relationship of DNA replication timing to different forms of human mutation and variation.</a> Am J Hum Genet. 91 (6), 1033-1040 (2012).</li><li>Rhind, N., Gilbert, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+replication+timing.">DNA replication timing.</a> Cold Spring Harb Perspect Biol. 5 (8), a010132 (2013).</li><li>Koren, 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=Genetic+variation+in+human+DNA+replication+timing.">Genetic variation in human DNA replication timing.</a> Cell. 159 (5), 1015-1026 (2014).</li></ol>

CNR-ToR kan uitgevoerd worden in principe op elke eukaryote prolifererende celpopulatie die kunnen worden gedeeld door FACS op S en G1 fasen (beoordeeld door Rhind N. en Gilbert 20 DM). De hier beschreven methode is aangepast voor zoogdiercellen met een genoomgrootte van ~ 3 Gb zoals mens en muis. Kleine veranderingen in de CNR-ToR protocol (in celpreparaat en sequentiebepaling diepte) nodig, om aan te passen aan andere eukaryoten. Aandacht moet worden besteed aan het verzamelen van voldoende hoe...

Zoogdier DNA replicatie strak geregeld om de juiste replicatie van elk chromosoom precies één keer te verzekeren tijdens de celcyclus. Replicatie gebeurt op basis van een sterk gereguleerde order - meerdere grote genomische regio&#39;s (~ Mb) te herhalen aan het begin van de S-fase (begin repliceren domeinen), terwijl andere genomische regio&#39;s later bootsen midden of late S-fase (midden en late replicerende domeinen) 1. De meeste genoom repliceert tegelijk in alle weefsels (constitutieve ToR domeinen), terwijl 30% - 50% van het genoom verandert ToR tussen weefsels 2, tijdens differentiatie 3, 4 en in mindere mate ook bij kanker transformatie 5 . Bovendien bepaalde genoomgebieden repliceren asynchroon 6, 7, 8, namelijk er is een verschilin de ToR tussen de twee allelen. ToR correleert met vele genomische en epigenomisch functies, waaronder transcriptie niveaus, GC-gehalte, chromatine staat, gen dichtheid, enz. 1, 9. ToR wordt ook geassocieerd met mutatie tarieven en types 10, 11 en daarom niet verwonderlijk, worden verstoringen van de replicatie-programma verband gebracht met kanker 12, 13. Het causale verband tussen de taakomschrijving en chromatine structuur is nog niet begrepen. Het is mogelijk dat geopend chromatine vergemakkelijkt vroege replicatie. Echter, een alternatief model suggereert dat het chromatine gemonteerd tijdens de replicatie en de verschillende chromatine regulatoren bij het begin en het einde van S-fase leidt tot differentiële verpakking van vroeg en laat replicerende gebieden 1, 14 </sup&gt;. We hebben onlangs aangetoond dat de ToR geeft vorm aan de GC-gehalte van invloed zijn op de aard van de mutaties die zich voordoen in verschillende genomische regio&#39;s 11. Fluorescentie in situ hybridisatie (FISH) is de belangrijkste methode voor het meten ToR op individueel loci. Het is eenvoudig uitgevoerd door het tellen van het percentage S-fase cellen die één FISH signalen versus percentage doubletten voor een bepaald allel 15, 16 vertonen. Een alternatieve methode bestaat uit het labelen van de puls DNA met BrdU, sorteren van cellen op basis van hun DNA inhoud aan verschillende tijdstippen langs S, immunoprecipitatie DNA met BrdU en controleren van de overvloed aan DNA neergeslagen met 17 qPCR. Genomische ToR mapping kan worden bereikt op twee manieren. De eerste methode is een genomische versie van de BrdU-IP hierboven beschreven, waarin de kwantificering van de hoeveelheidvan geprecipiteerd DNA in elke fractie gelijktijdig uitgevoerd voor het volledige genoom tot hybridisatie aan microarrays of diepe sequencing. De tweede methode, CNR-referentiekader, is gebaseerd op het meten van het aantal kopieën van elk genomisch gebied van S-fase cellen en het normaliseren van het DNA-gehalte in G1 cellen. Bij deze werkwijze worden de cellen gesorteerd door FACS in niet-replicerende (G1-fase) en repliceren (S-fase) groepen (Figuur 1). Cellen in G1 hetzelfde aantal kopieën in alle genomische regio en zullen hun DNA inhoud hetzelfde zijn. Anderzijds, het DNA kopieaantal in S afhankelijk van het referentiekader, sinds begin replicerende regio onderging replicatie in de meeste cellen en dus hun DNA-inhoud wordt verdubbeld, terwijl laat replicerende gebieden nog in de meeste cellen gerepliceerd en dus hun DNA inhoud analoog is aan die van G1 cellen. Vandaar de S om G1-verhouding van DNA-gehalte is een indicatie van het mandaat. De hoeveelheid DNA per genomisch gebied wordt gemeten door hybridisatie metmicroarrays of door diepe sequencing 2, 8. De voordelen van het CNR-ToR methode zal verder worden besproken. Dit document beschrijft de CNR-Tor methode voor het genoom ToR in kaart brengen, zoals beschreven in figuur 2. Het artikel bespreekt de fijne details van het gehele proces van het verzamelen van de cellen tot de fundamentele analyse van de resultaten en het creëren van genomische ToR kaarten. De in dit document beschreven protocol is met succes uitgevoerd op verschillende celtypen in kweek. Toekomstige verbeteringen van dit protocol kan leiden tot het in kaart brengen van het referentiekader in vivo en in zeldzame celtypen.

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			<td height="21" style="height:21px;width:339px;">PBS</td>
			<td style="width:157px;">BI (Biological Industries)</td>
			<td style="width:119px;">02-023-1A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA</td>
			<td>BI (Biological Industries)</td>
			<td>03-052-1B</td>
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		<tr height="21">
			<td height="21" style="height:21px;">15ml conical tube</td>
			<td>Corning</td>
			<td align="right">430790</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5ml Polystyrene round Bottom tube with cell strainer cap&#160;</td>
			<td>BD-Falcon</td>
			<td align="right">352235</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Gadot</td>
			<td>64-17-5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAse-A 10mg/ml</td>
			<td>Sigma</td>
			<td>R4875</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propidiom iodide 1mg/ml</td>
			<td>Sigma</td>
			<td>P4170</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">parafilm</td>
			<td>Parafilm</td>
			<td>PM-996</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5ml DNA LoBind Eppendorf tubes&#160;</td>
			<td>Eppendorf</td>
			<td align="right">22431021</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Sigma</td>
			<td>A7906</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.7ml MaxyClear tube&#160;</td>
			<td>Axygen</td>
			<td>MCT-175-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">magnetic beads - Agencourt AMPure XP&#160;</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ultrasonicator</td>
			<td>Covaris</td>
			<td>M-series&#160; -530092</td>
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		<tr height="21">
			<td height="21" style="height:21px;">50 &#181;l microTUBE AFA Fiber Screw-Cap 6x16mm</td>
			<td>Covaris</td>
			<td align="right">520096</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Qubit fluorometer</td>
			<td>Invitrogen</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit dsDNA High Sensitivity (HS) Assay Kit</td>
			<td>Invitrogen</td>
			<td>Q32854</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electrophoresis.2200 Tape station system</td>
			<td>Agilent</td>
			<td>D1000 ScreenTape</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Seqeuncing - Illumina NextSeq system</td>
			<td>Illumina</td>
			<td>SY-415-1001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dneasy kit for DNA purification</td>
			<td>Qiagen</td>
			<td align="right">69504</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PureProteom Magnetic Stand</td>
			<td>Millipore</td>
			<td>LSKMAGS08</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-BrdU/FITC</td>
			<td>DAKO</td>
			<td>F7210</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS sorter</td>
			<td>BD</td>
			<td>FACSARIA III</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS software</td>
			<td>BD</td>
			<td>FACSDiva v 8.0.1</td>
		</tr>
	</tbody>
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genome-wide determination of mammalian replication timing by dna content measurement

Replicatie van het genoom plaatsvindt tijdens de S fase van de celcyclus in een sterk gereguleerd proces dat de getrouwheid van DNA-duplicatie zorgt. Elk genomisch gebied wordt gerepliceerd op een specifieke tijd gedurende de S-fase door de gelijktijdige activering van verschillende oorsprongen van replicatie. Tijd van replicatie (ToR) correleert met vele genoom en epigenetische kenmerken en is gekoppeld aan mutatie tarieven en kanker. Het begrijpen van de volledige genomische uitzicht op de replicatie-programma, in gezondheid en ziekte is een belangrijke toekomstige doel en uitdaging. Dit artikel beschrijft in detail de &quot;Copy Number Verhouding S / G1 voor het in kaart brengen van genomische Time of Replication&quot; methode (hierin genoemd: CNR-ToR), een eenvoudige benadering van de genoomwijde ToR van zoogdiercellen kaart te brengen. De werkwijze is gebaseerd op het kopienummer verschillen tussen de S-fase cellen en G1-fase cellen. De CNR-ToR werkwijze wordt uitgevoerd in 6 stappen: 1. Bereiding van cellen en kleuring met propidiumjodide (PI); 2. Sorting G1 en S-fase cellen met behulp van fluorescentie-geactiveerde celsortering (FACS); 3. DNA-zuivering; 4. Sonicatie; 5. voorbereiding en sequencing Bibliotheek; en 6. Bioinformatica analyse. De CNR-ToR methode is een snelle en eenvoudige aanpak die resulteert in gedetailleerde replicatie kaarten.

Een typische ToR kaart wordt weergegeven in figuur 3 voor muis embryonale fibroblasten (MEF). Deze figuur toont het analyseproces aangezien toont zowel de punten die de genormaliseerde S / G1 ratio voor afzonderlijke vensters (stap 8,3) zijn, en de lijn die door de kubische smoothing en interpolatie (stap 8,5). Dergelijke kaarten vast te leggen voor de organisatie van de replicatie-programma, dat is een lappen...

Watch this Scientific Journal Video about Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement at JoVE.com

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

Genoom-brede analyse van zoogdieren Replication Timing door DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

The overall goal of this procedure, is to map the genome-wide replication timing program of a cell of choice. This method can help answer key questions in the replication field. Such as, how the genomic replication program changes in health and disease.The main advantage of this technique is that it is a fast and simple way to map replication timing in various cells, and conditions. The man writing the fax procedure would be Dan Lehmann, a fax expert in The Faculty of Medicine. This procedure usually starts with at least one to two times ten to the sixth fast growing unsynchronized cells.For adherent cells, aspirate and wash each plate with three milliliters of PBS without calcium of magnesium. Discard the PBS and add one milliliter of commercial Trypsin-EDTA to each plate. Incubate the cells for five minutes, at 37 degrees Celsius, until they detach.Add three milliliters of culture media to neutralize the Trypsin and collect the cells in a 15 milliliter conical tube or a five milliliter polystyrene tube. Keep the cells on ice. To begin theisprocedure, centrifuge the cells at 300 x G for five minutes at four degrees Celsius.Aspirate and wash the cells with one milliliter of cold PBS. Then, spin down the cells. After the second wash, remove the PBS and resuspend the cells in 250 microliters of cold PBS.While gently vortexing the tube, slowly add drop wise 800 microliters of minus 20 degree Celsius, 100 percent high purity ethanol. This leads to our final ethanol concentration of 70 to 80 percent. Incubate the cells on ice for 30 minutes.Begin this procedure by centrifuging the fixed cells at 500 x G for ten minutes at 4 degrees Celsius. Aspirate the supernatant carefully and wash the cells twice with one milliliter of cold PBS. Spin down after each wash.After the second wash, aspirate the supernatant and resuspend each cell with the following PI mixture. One milliliter of PBS containing five microliters of ten milligram per milliliter RNAas, and 50 microliters of one milligram per milliliter propidium iodide. Filter the sample through a 35 micrometer mesh into a 5 milliliter polystyrene tube.Seal the tube with parafilm. Incubate at room temperature for 15 to 30 minutes. Keep the samples in the dark as propidium iodide is light sensitive.The cells will be sorted using a fax machine. Use the 561 nanometer laser to differentiate cells based on their propidium iodide intensity. For optimal results, use the smallest nozzle recommended for the specific cell size.For most cells, it is 85 micrometers. Use a steady, slow flow. Usually up to 300 to 500 events per second.Using Gating, discriminate dead cells and subcellular debris by plotting FCS versus SSC. From the viable cells, discriminate doublets by plotting SSC width versus SSC height, followed by an FFC width versus FFC height plot, and a propidium iodide width versus propidium iodide height plot. For the viable signal cells, draw a histogram of the propidium iodide area intensity, which represents the DNA content of the cells.Sort cells in cold conditions into G1 and S phases. Gating for S should be wide and intrude into the G1 and G2 phases. While G1 gating should be narrow and as far from S as possible.For come cell types, it may be hard to get a nice cell cycle distribution. It is most critical to take the G1 phase without S phase cells. Remove tubes with sorted cells from the fax machine.Keep tubes on ice following the sort. Following the cell sorting, the DNA for each sample is purified using a commercial DNA purification kit following the manufacturer's directions. Next, sheer the DNA with a focused ultrasonicator to an average target peak size of 250 base pairs.Verify the size of the sheer DNA by electroforesis. The recommended size distribution is 200 to 700 base pairs with a peak at about 250 base pairs. Subsequently, the library is prepared, sequenced, and analyzed as described in the text protocol.A time of replication map for the entire chromosome one from mouse embryonic fibroblasts is shown. With a closer look at a region of approximately 50 megabases. The dots represent the normalized S to G1 ratio for individual windows, and the solid line represents the results from the cubic smoothly and interpolation.High S to G1 values corresponds to early replication, whereas low values correspond to late replication. The red arrows point to examples of constant time of replication regions, and the green arrows point to temporal transition regions. This figure shows the reproducibility of time of replication maps between triplicates of mouse embryonic fibroblasts, compared to triplicates of mouse pre-B cells in the same 18 megabase region on mouse chromosome one.The orange arrows, point to examples of differential regions between the cell lines. A heat map of the spearman correlations between the six samples further confirm the results. Once mastered, this technique from the collection of cells to the generation of maps, can be completed in one week if performed properly.After watching this video, you should have a good understanding of how to generate time of replication genomic maps for growing cells.

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Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Kinetiek van Achterblijvende-streng DNA-synthese<em&gt; In Vitro</em&gt; Door de bacteriofaag T7 Replication Eiwitten

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

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

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

Genoom-wide mapping van eiwit-DNA-interacties met ChEC-seq in<em&gt; Saccharomyces cerevisiae</em

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

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Bepaling van de optimale chromosomale locatie (s) voor een DNA Element in Escherichia coli met behulp van een nieuwe benadering van Transposon-gemedieerde

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Toewijzing genoom-brede toegankelijk chromatine in primaire menselijke T lymfocyten door ATAC-Seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts

Bepalen van genoom-brede Transcript verval tarieven in delende en zwakke menselijke fibroblasten

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ...

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

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

Generatie van genoom-brede chromatine conformatie Capture bibliotheken uit strak geënsceneerde vroege Drosophila embryo 's

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

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Profilering van DNA replicatie Timing met behulp van de zebravis als een modelsysteem In Vivo

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

Promotor Capture Hi-C: High-resolution, genoom-brede profilering van promotor interacties

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

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

Een eenvoudige, snelle en kwantitatieve bepaling aan maatregel reparatie van DNA-proteïne Crosslinks op plasmiden Transfected in zoogdiercellen

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 ...