Diese Arbeit wurde unterstützt durch Zuschuss NIH GM117446 an A.B

1. Vorbereitung der Zellen für Flow Cytometry sortieren
<ol><li>15 mL Reagenzgläsern, 8 mL YEPD Brühe enthält, so dass die Kulturen eine Dichte von 5 x 106 bis 1,5 x 107 Zellen pro mL nach Übernachtung Wachstum erreichen zu impfen (siehe Diskussion Hinweis 1).</li>
	<li>Spin-down Hefezellen (1.400 x g, bei Raumtemperatur oder 4 ° C) in eine Schraubkappe Zentrifugenröhrchen 15 mL für 5 min, Zellen in 1,5 mL 70 % igem Ethanol und Transfer nach einem 1,6 mL Microfuge Rohr, lassen diese Sit für 1 h bei Raumtemperatur oder mindestens 3 h auf Eis (Aufschwemmen können gespeichert werden auf unbestimmte Zeit bei 4 ° C) (siehe Diskussionspunkt (2)).</li>
	<li>Spin-down Hefezellen in die Microfuge (14.000 x g, 4 ° C) für 1 min in 1 mL 50 mM Natriumcitrat, pH 7,2, Spin-down (14.000 x g, 4 ° C) für 1 min aufzuwirbeln, Aspirieren überstand, Aufschwemmen in 1 mL 50 mM Natriumcitrat, pH 7,2 , mit 0,25 mg/mL RNAse-Lösung für 1 h bei 50 ° C oder über Nacht bei 37 ° C in Hitze Block oder Wasserbad.</li>
	<li>Fügen Sie 50 µL Proteinase K-Lösung (20 mg/mL Nukleinsäuretablette in 10 mM Tris pH 7.5, 2 mM CaCl2, 50 % Gewicht, Volumen Glycerin) und 1 h bei 50 ° c inkubieren</li>
	<li>Spin-down Zellen (14.000 x g, 4 ° C), Aufschwemmen Zellen in 1 mL 50 mM Natriumcitrat, pH 7,2, spin-down (14.000 x g, 4 ° C), überstand mit Vakuum anzusaugen, Aufschwemmen in 1 mL 1 µM grün Nukleinsäure-Stamm Nukleinsäuretablette in 50 mM Natriumcitrat , pH 7,2 und in der Dunkelheit für 1 h bei Raumtemperatur inkubieren, beschallen 2 X für 2 s (Ausgangsleistung 2 Watt).</li>
</ol>(2) Zellsortierung
<ol><li>Mit einer Zelle Sorter, sortieren Zellen nach DNA-Gehalt in Phasen G1, S, "frühen G2" und "späten G2", sammeln von mindestens 1,6 Millionen haploiden Zellen von jedem Zellzyklus-Phase wie in Abbildung 1 (siehe Diskussionspunkt (3)).</li>
	<li>Zellen auf Microfuge Röhren übertragen und spin-down Zellen für 20 min bei 14.000 x g bei 4 ° C in der Microfuge und aspirieren überstand mit Vakuum (das Pellet kann bei-20 ° C eingefroren auf unbestimmte Zeit gespeichert werden, siehe Diskussion Punkt (4)).</li>
</ol>3. DNA-Extraktion und die Vorbereitung der Sequenzierung Bibliotheken
Hinweis: Die folgenden Schritte basieren auf "Protokoll I" in der Hefe Genomic DNA Extraktion Kit (siehe Tabelle der Materialien).
<ol><li>Fügen Sie 120 µL "Verdauung Puffer," eine proprietäre Puffer, der Hefe lytische Enzyme verdauen die Zellwand und 5 µL Hefe lytische Enzym, Aufschwemmen durch aufschütteln und Inkubation bei 37 ° C für 40-60 min ermöglicht.</li>
	<li>Fügen Sie 120 µL "Lysis Buffer," eine proprietäre Puffer, Waschmittel und hart für 10-20 s Wirbel enthält.</li>
	<li>Fügen Sie 250 µL Chloroform - mischen für 1 min.</li>
	<li>Mit Höchstgeschwindigkeit in einem Microfuge für 2 min drehen.</li>
	<li>Laden Sie den Überstand auf der DNA-Bindung Spalte und Zentrifuge mit maximaler Geschwindigkeit in eine Microfuge für 1 min.</li>
	<li>Fügen Sie 300 µL "DNA waschen Puffer", ein proprietäres Puffer, Ethanol und Zentrifuge für 1 min bei maximaler Geschwindigkeit in eine Microfuge enthält. Verwerfen der Durchströmung, 300 µL Waschpuffer DNA hinzufügen und wiederholen Sie den Waschvorgang zu.</li>
	<li>Die Spin-Spalte auf ein frisches Microfuge Rohr übertragen, fügen 60 µL Wasser (nicht TE) - 1-3 min warten und dann Zentrifugieren für 10 s, um die DNA zu eluieren.</li>
	<li>Konzentration durch Zugabe von 5 µL DNA zu 195 µL DsDNA-hohe Empfindlichkeit-Reagenz, das verdünnt 1: 200 in DsDNA-hohe Empfindlichkeit-Puffer und dann Fluoreszenz-Maßnahme in einem Fluorometer, mit 10 µL der mitgelieferten Standards für die Kalibrierung wurde zu messen; zwischen 10 und 50 ng der DNA Gesamtausbeute erwarten.</li>
	<li>Beschallen (Peak Vorfall macht 450, Einschaltdauer 30 % Zyklen pro Burst 200, Behandlung Zeit 60 s, Wasserstand 6) DNA in 250-350 bp Fragmente aufzubrechen.</li>
	<li>Bereiten Sie eine Bibliothek mit DNA-Probe Vorbereitungssatz und es (mindestens 10 Millionen 50 bp Einend-gelesen) auf die DNA-Sequenzierung Instrument im Schnellmodus-Sequenz (vgl. Diskussion (5)).</li>
</ol>4. Analyse der Daten und Erstellung von Replikation Sequenzierung Profile
<ol><li>Ausrichten von Saccharomyces Cerevisiae sacCer3 Genom mit Gsnap12 -Sequenzen (siehe Diskussionspapier (6)).</li>
	<li>Bestimmen Sie, dass pro Basenpaar tiefen mit Bedtools "GenomeCoverageBed" Software13lesen.</li>
	<li>Artifizielle Spikes in Lesen Sie tiefen zu entfernen, falls vorhanden (siehe Diskussionspapier (7)).</li>
	<li>Glatten Tiefen von Chromosom mit Mittelstreifen in eine 20 kb Schiebefenster mit "RunMean" Funktion in R Paket "CaTools" gelesen (siehe Diskussionspapier (8)).</li>
	<li>Plot gelesen tiefen in S Phase Anfang G2 und späten G2 als prozentualer Anteil vollständig replizierten lesen Sie tiefen (siehe Diskussionspapier (9)).</li>
</ol>

<ol>
	<li>Fragkos, M., Ganier, O., Coulombe, P., Mechali, 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+origin+activation+in+space+and+time.">DNA replication origin activation in space and time.</a> Nat Rev Mol Cell Biol. 16, 360-374 (2015).</li><li>Santocanale, C., Diffley, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mec1-+and+Rad53-dependent+checkpoint+controls+late-firing+origins+of+DNA+replication.">A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication.</a> Nature. 395, 615-618 (1998).</li><li>Brewer, B. J., Fangman, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+replication+fork+barrier+at+the+3'+end+of+yeast+ribosomal+RNA+genes.">A replication fork barrier at the 3' end of yeast ribosomal RNA genes.</a> Cell. 55, 637-643 (1988).</li><li>Raghuraman, M. K., 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=Replication+dynamics+of+the+yeast+genome.">Replication dynamics of the yeast genome.</a> Science. 294, 115-121 (2001).</li><li>Muller, C. A., Nieduszynski, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+replication+timing+reveals+global+and+local+regulation+of+replication+origin+activity.">Conservation of replication timing reveals global and local regulation of replication origin activity.</a> Genome Res. 22, 1953-1962 (2012).</li><li>Koren, A., Soifer, I., Barkai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRC1-dependent+scaling+of+the+budding+yeast+DNA+replication+timing+program.">MRC1-dependent scaling of the budding yeast DNA replication timing program.</a> Genome Res. 20, 781-790 (2010).</li><li>Lang, G. I., Murray, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+rates+across+budding+yeast+chromosome+VI+are+correlated+with+replication+timing.">Mutation rates across budding yeast chromosome VI are correlated with replication timing.</a> Genome Biol Evol. 3, 799-811 (2011).</li><li>Durkin, S. G., Glover, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+fragile+sites.">Chromosome fragile sites.</a> Annu Rev Genet. 41, 169-192 (2007).</li><li>Dillon, L. W., Burrow, A. A., Wang, Y. 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+instability+at+chromosomal+fragile+sites+in+cancer.">DNA instability at chromosomal fragile sites in cancer.</a> Curr Genomics. 11, 326-337 (2010).</li><li>Widrow, R. J., Hansen, R. S., Kawame, H., Gartler, S. M., Laird, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Very+late+DNA+replication+in+the+human+cell+cycle.">Very late DNA replication in the human cell cycle.</a> Proc Natl Acad Sci U S A. 95, 11246-11250 (1998).</li><li>Foss, E. 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=SIR2+suppresses+replication+gaps+and+genome+instability+by+balancing+replication+between+repetitive+and+unique+sequences.">SIR2 suppresses replication gaps and genome instability by balancing replication between repetitive and unique sequences.</a> Proc Natl Acad Sci U S A. 114, 552-557 (2017).</li><li>Wu, T. D., Reeder, J., Lawrence, M., Becker, G., Brauer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GMAP+and+GSNAP+for+Genomic+Sequence+Alignment:+Enhancements+to+Speed,+Accuracy,+and+Functionality.">GMAP and GSNAP for Genomic Sequence Alignment: Enhancements to Speed, Accuracy, and Functionality.</a> Methods Mol Biol. 1418, 283-334 (2016).</li><li>Quinlan, A. R., Hall, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BEDTools:+a+flexible+suite+of+utilities+for+comparing+genomic+features.">BEDTools: a flexible suite of utilities for comparing genomic features.</a> Bioinformatics. 26, 841-842 (2010).</li><li>Langmead, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aligning+short+sequencing+reads+with+Bowtie.">Aligning short sequencing reads with Bowtie.</a> Curr Protoc Bioinformatics. , 17 (2010).</li><li>Li, H., Durbin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+accurate+short+read+alignment+with+Burrows-Wheeler+transform.">Fast and accurate short read alignment with Burrows-Wheeler transform.</a> Bioinformatics. 25, 1754-1760 (2009).</li><li>Kellis, M., Birren, B. W., Lander, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proof+and+evolutionary+analysis+of+ancient+genome+duplication+in+the+yeast+Saccharomyces+cerevisiae.">Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae.</a> Nature. 428, 617-624 (2004).</li><li>Goodwin, S., McPherson, J. D., McCombie, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coming+of+age:+ten+years+of+next-generation+sequencing+technologies.">Coming of age: ten years of next-generation sequencing technologies.</a> Nat Rev Genet. 17, 333-351 (2016).</li></ol>

Die Autoren haben nichts preisgeben.

Während diese Technik robust ist und relativ geradlinig, besondere Aufmerksamkeit werden, an folgende gewidmet sollte:
(1) Wir empfehlen, mindestens 12 h Kulturen wachsen, bevor sie Log-Phase erreichen, da Unterschiede im Zellzyklus Distributionen zu manifestieren, wenn Kulturen geerntet werden, nach dem Erreichen der gewünschten Dichte nur 4 h nach der Inokulation. Unsere Annahme ist, dass eine Zellzyklus-Distribution, die ein relativ stabiles Gleichgewicht erreicht hat besser als eine Vert...

Eukaryotische Genom Replikation wird eingeleitet, an mehreren diskreten Standorten, genannt Ursprünge der Replikation, von welchen Replikation Gabeln in beide Richtungen (rezensiert in Fragkos Et Al., 20151) gehen. Ursprünge variieren in ihren Zeitplan und die Effizienz der Zündung und verschiedene Techniken wurden entwickelt, um Replikation Herkunft Aktivitäten überwachen und die Ursachen dieser Unterschiede zu erhellen. Die Aktivität der einzelnen Ursprünge kann aus einsträngiger DNA2, welche Formulare rund um aktive Ursprung abgeleitet werden oder mithilfe von 2D Gelelektrophorese, um spezielle Replikations Zwischenprodukte3zu überwachen, können beide mit erkannt werden radioaktiven Sonden. Beide Techniken sind leichter in S. Cerevisiae als in Säugerzellen, angewendet, da Ursprünge auf bestimmten bekannten Sequenzen in der ehemaligen beschränkt sind. Mit dem Aufkommen von Microarrays wurde es möglich, die Herkunft weltweit brennen zu beurteilen. Dies geschah zunächst durch Kennzeichnung DNA mit schweren Isotopen, Freisetzung von Zellen aus einem G1 in mittlerer mit leichten Isotope und dann die Bildung von schwer-leicht-Hybrid DNA in das Genom4Überwachung. Die Einführung von Hochdurchsatz-Sequenzierung erlaubt ähnliche genomweite Überwachung der Herkunft Aktivität ohne das Erfordernis einer teuren Isotopen Kennzeichnung. Zellen wurden in einem Durchflusszytometer laut DNA-Gehalt und ihre DNA ausgesetzt Tiefe Sequenzierung sortiert. Da Sequenz Abdeckung von 1 X bis 2 X im Laufe der S-Phase verläuft, kann relative Replikation Timing durch Vergleich lesen Sie Tiefen der Zellen in der S-Phase, die in G1 oder G25,6bewertet werden. Diese Techniken, vor allem Hefe, führte zu einem tieferen Verständnis der wie DNA-Sequenz, Chromatinstruktur und DNA-Replikation Proteine Herkunft Timing und Effizienz regulieren.
Treue Übertragung der genetischen Information während der Zellproliferation erfordert nicht nur erfolgreiche Initiation der DNA-Replikation, die stattfindet am Ursprung, sondern auch erfolgreichen Abschluss der Replikation, die auftritt, wo Replikations-Gabeln treffen. Wie die Initiation der Replikation variiert der Zeitpunkt der Fertigstellung der Replikation des Genoms mit bestimmten Regionen bleiben bis spät in den Zellzyklus Langzeittransaktionen. Solche Regionen enthalten Sequenzen oder Chromatin Strukturen, die DNA-Polymerasen behindern oder möglicherweise besonders aktive Replikation Ursprünge fern. Spät repliziert Regionen kann als fragile Sites, manifestieren sich die chromosomalen Bruch und höhere Mutationsraten zugeordnet sind, und haben bei Krebs und Altern7,8,9in Verbindung gebracht. Allerdings hat unser Verständnis von wo und wie dies geschieht trotz der Bedeutung der ordnungsgemäßen Abschluss der DNA-Replikation in der Instandhaltung der Genomstabilität weit hinter der Initiation der Replikation hinterher. Und während einzelner Gene, deren späte Replikation mit Krankheit assoziiert wurde, mit z. B. qPCR10, globale Studien zur Aufklärung der Standorte und die zugrunde liegenden Ursachen der letzten Replikation gefehlt untersucht worden. Hier beschreiben wir eine Technik, die wir uns beziehen, um als "G2 Seq" Wir Durchflusszytometrie mit Hochdurchsatz-Sequenzierung verbinden Aufschluss über die Vollendung des Genoms Replikation in Hefe-11. Mit geringfügigen Änderungen kann dieses Protokoll auf alle Zellen angepasst werden, die Strömung nach DNA-Gehalt sortiert werden können.

<table width="722">
	<tbody>
		<tr height="21">
			<td height="21" style="width:301px;height:21px;">YeaStar Genomic DNA kit</td>
			<td style="width:135px;">Genesee Scientific</td>
			<td style="width:119px;">11-323</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:301px;height:21px;">1 &#181;M SYTOX Green</td>
			<td style="width:135px;">ThermoFisher</td>
			<td style="width:119px;">57020</td>
			<td>resuspend in 50 mM sodium citrate, pH 7.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:301px;height:21px;">50 mM sodium citrate, pH 7.2</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="147">
			<td height="147" style="width:301px;height:147px;">RNase solution (0.25 mg / mL)</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">R6513</td>
			<td style="width:167px;">0.25 mg / mL RNaseA resuspended in 50 mM sodium citrate, pH 7.2, boil RNase solution for 10 minutes before the first use only, and from then on store at -20&#176;</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:301px;height:42px;">proteinase K solution (20 mg / mL)</td>
			<td style="width:135px;">ThermoFisher / Invitrogen</td>
			<td style="width:119px;">25530-015</td>
			<td>resuspend in 10 mM Tris, pH 7.5, 2 mM CaCl2, 50% glycerol, store at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Model 50 Sonic Dismembrator</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td>FB50A220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:301px;height:21px;">BD Biosciences FACSAria II</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>644832</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:301px;height:21px;">Zymo-spin III columns</td>
			<td style="width:135px;">Zymo Research</td>
			<td style="width:119px;">C1005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:301px;height:21px;">Qubit dsDNA HS Assay Kit</td>
			<td style="width:135px;">ThermoFisher</td>
			<td style="width:119px;">Q32851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:301px;height:21px;">Qubit 3.0 Fluorometer</td>
			<td style="width:135px;">ThermoFisher</td>
			<td style="width:119px;">Q33216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Covaris Model LE220 Focused-Ultrasonicator</td>
			<td style="width:135px;">Covaris</td>
			<td style="width:119px;">500219</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:301px;height:21px;">Illumina TruSeq DNA LT Sample Prep Kit</td>
			<td style="width:135px;">Illumina</td>
			<td>15026486</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:301px;height:21px;">Illumina HiSeq 2500 instrument</td>
			<td style="width:135px;">Illumina</td>
			<td>SY&#8211;401&#8211;2501&#160;</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:301px;height:63px;">gsnap alignment software</td>
			<td style="width:135px;">open source software / Genentech</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">http://research-pub.gene.com/gmap</td>
		</tr>
	</tbody>
</table>

g2-seq: a high throughput sequencing-based technique for identifying late replicating regions of the genome

Zahlreiche Techniken wurden entwickelt, um den Fortschritt der DNA-Replikation durch die S-Phase des Zellzyklus folgen. Die meisten dieser Techniken sind in Richtung Aufklärung des Standortes und der Zeitpunkt der Einleitung des Genoms Vervielfältigung, anstatt seine Vollendung verwiesen worden. Es ist jedoch wichtig, dass wir Regionen des Genoms, die letzten Replikation abgeschlossen sind verstehen, weil diese Regionen erhöhten chromosomalen Bruch und Mutation leiden, und sie im Zusammenhang mit Krankheit und Alterung wurden. Hier beschreiben wir, wie wir eine Technik, die verwendet wurde, um die Replikation Einleitung um stattdessen diesen Regionen des Genoms letzte vollständige Replikation identifizieren überwachen ausgedehnt haben. Dieser Ansatz basiert auf einer Kombination der Durchflusszytometrie und Hochdurchsatz-Sequenzierung. Obwohl dieser Bericht konzentriert sich auf die Anwendung dieser Technik auf Hefe, kann der Ansatz mit beliebigen Zellen verwendet werden, die in einem Durchflusszytometer laut DNA-Gehalt sortiert werden können.

Wir haben wie oben beschrieben vor spät replizierenden Standorte in der angehenden Hefe verwendet. Tests dieser Ansatz mit einer bekannten späten replizierende Region auf ein künstliches Chromosom erwies sich die Technik präzise und zuverlässig sein. Unsere Ergebnisse haben auch gezeigt, die biologische Bedeutung der rechtzeitigen Abschluss der Replikation zeigen, dass eine spätere replizierende Region auf Chromosom 7, die wir identifiziert als späte Replikation auf der Grundlage u...

Watch this Scientific Journal Video about G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome at JoVE.com

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

G2-Seq: Ein hoher Durchsatz Sequenzierung-basierten Methode zur Identifizierung von spät repliziert Regionen des Genoms

Wir beschreiben eine Technik Durchflusszytometrie und Hochdurchsatz-Sequenzierung zu spätere replizierende Regionen des Genoms zu identifizieren, zu kombinieren.

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

g2-seq-high-throughput-sequencing-based-technique-for-identifying

Research

JoVE Journal

Genetics

5.7K Aufrufe.  Fred Hutchinson Cancer Research Center. Wir beschreiben eine Technik Durchflusszytometrie und Hochdurchsatz-Sequenzierung zu spätere replizierende Regionen des Genoms zu identifizieren, zu kombinieren.

Serie: G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Ein Roboter-Plattform für Hochdurchsatz-Isolierung von Protoplasten und Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Forschung

Genetik

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Zuordnung von RNA-RNA-Wechselwirkungen unter Verwendung von biotinyliertem Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

Retroviral Scanning: Mapping MLV Integration Sites zur Definition zellspezifischer Regulierungsregionen

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

Erzeugung von nativen Chromatin Immunopräzipitation Sequenzierung Bibliotheken für Nukleosom Dichteanalyse

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

Robuste DNA-Isolierung und Hochdurchsatz-Sequenzierung Bibliotheksbau für Herbar

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

Embryo Mikroinjektion und Transplantation Technik für Nasonia Vitripennis Genom Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

Hochdurchsatz-Identifizierung von synergistischen Wirkstoffkombinationen durch die Überlappung2 Methode

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

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

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

Promotor Capture Hallo-c: Hochauflösende, genomweite Profilierung der Promoter Interaktionen

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

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

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

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

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

Hochdurchsatz-Identifizierung des Gens regulatorischen Sequenzen mit Next-Generation Sequenzierung der kreisförmigen Chromosom Konformation erfassen (4C-Seq)

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

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

Efficient genome engineering of Candida albicans is critical to understanding the pathogenesis and development of therapeutics. Here, we described a protocol to quickly and accurately edit the C. albicans genome using CRISPR. The protocol allows investigators to introduce a wide variety of genetic modifications including point mutations, insertions, and deletions.

CRISPR-vermittelten Genom-Bearbeitung von menschlichen pilzliche Erreger Candida albicans

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...