Questo lavoro è stato supportato da grant GM117446 NIH di A.B.

1. preparazione delle cellule per flusso Cytometry ordinamento
<ol><li>Inoculare provette da 15 mL contenente 8 mL di brodo YEPD tale che le culture raggiungono una densità di 5 x 106 a 1,5 x 107 cellule per mL dopo crescita durante la notte (vedi discussione Nota 1).</li>
	<li>Rotazione verso il basso le cellule di lievito (1.400 x g, a temperatura ambiente o a 4 ° C) in una provetta da centrifuga da 15 mL tappo a vite per 5 min, risospendere le cellule in 1,5 mL di etanolo al 70% e trasferirlo in una provetta di microfuge 1,6 mL, lasciando questo sedersi per 1 h a temperatura ambiente o almeno 3 h sul ghiaccio ( possono essere conservati a tempo indeterminato a 4 ° C) (Vedi punto di discussione (2)).</li>
	<li>Rotazione verso il basso le cellule di lievito in microfuge (14.000 x g, 4 ° C) per 1 min, risospendere in 1 mL 50 millimetri citrato di sodio, pH 7,2, spin-down (14.000 x g, 4 ° C) per 1 min, aspirare il surnatante e risospendere in 1 mL 50 millimetri citrato di sodio, pH 7,2 , contenente 0,25 mg/mL soluzione di RNAsi per 1 h a 50 ° C o una notte a 37 ° C in bagno di acqua o blocco di calore.</li>
	<li>Aggiungere 50 µ l di soluzione di proteinasi K (20 mg/mL risospesi in 10 mM Tris-HCl pH 7.5, 2mm CaCl2, 50% in peso di glicerolo di volume) e incubare per 1 h a 50 ° C.</li>
	<li>Rotazione verso il basso delle cellule (14.000 x g, 4 ° C), risospendere le cellule in 1 mL 50 millimetri di citrato di sodio, pH 7,2, rotazione verso il basso (14.000 x g, 4 ° C), aspirare il surnatante con vuoto, risospendere in 1 mL 1 µM verde acido nucleico ceppo risospeso in citrato di sodio 50 mM , pH 7.2 ed incubare al buio per 1 h a temperatura ambiente, Sonicare x 2 per 2 s ciascuno (2 watt di potenza di uscita).</li>
</ol>2. cella ordinamento
<ol><li>Usando un Classificatore celle, ordinare le celle in base al contenuto di DNA in fasi G1, S, "primi G2" e "fine G2", raccolta di cellule aploidi almeno 1,6 milioni da ogni fase del ciclo cellulare come in Figura 1 (Vedi punto di discussione (3)).</li>
	<li>Trasferire le cellule microfuge provette e spin giù cellule per 20 min a 14.000 x g a 4 ° C nel microfuge e aspirare il surnatante con vuoto (il pellet può essere conservato congelati a-20 ° C a tempo indeterminato; veda discussione punto (4)).</li>
</ol>3. DNA estrazione e preparazione di sequenziamento librerie
Nota: La procedura seguente si basata su "protocollo I" nell'estrazione del DNA genomico di lievito Kit (Vedi Tabella materiali).
<ol><li>Aggiungere 120 µ l di "Buffer di digestione," un buffer proprietario che consente degli enzimi litici di lievito a digerire la parete cellulare e 5 µ l di enzima litico lievito, risospendere nel vortex e incubare a 37 ° C per 40-60 min.</li>
	<li>Aggiungere 120 µ l di tampone di Lisi"," un buffer proprietario che contiene detergente e vortice dura per 10-20 s.</li>
	<li>Aggiungi 250 cloroformio µ l - Mescolare accuratamente per 1 min.</li>
	<li>Girare alla massima velocità in un microfuge per 2 min.</li>
	<li>Caricare il surnatante nella colonna associazione DNA e centrifugare in un microfuge alla massima velocità per 1 min.</li>
	<li>Aggiungere 300 µ l di "DNA lavaggio Buffer," un buffer proprietario che contiene etanolo e centrifugare per 1 min in un microfuge alla massima velocità. Scartare il flusso attraverso, aggiungere 300 µ l di tampone di lavaggio di DNA e ripetere il processo di lavaggio.</li>
	<li>Trasferire la colonna di spin in una provetta di microfuge fresco, aggiungere 60 µ l di acqua (non TE) - attendere 1-3 min e poi Centrifugare per 10 s per eluire il DNA.</li>
	<li>Misurare la concentrazione aggiungendo 5 µ l di DNA a 195 µ l di reagente di dsDNA ad alta sensibilità che è stato diluito 1: 200 in buffer di dsDNA ad alta sensibilità e quindi misura fluorescenza in un fluorimetro, utilizzando 10 µ l di dotazione standard per la calibrazione; si aspettano tra 10 e 50 ng di rendimento totale del DNA.</li>
	<li>Sottoporre ad ultrasuoni (picco incidente potenza 450, fattore di utilizzo del 30%, cicli al Burst 200, trattamento tempo 60 s, livello acqua 6) per spezzare il DNA in frammenti di 250-350 bp.</li>
	<li>Preparare una libreria utilizzando il kit di preparazione del campione del DNA e la sequenza è (almeno 10 milioni di 50 bp singolo fine letture) dello strumento di sequenziamento del DNA in modalità rapida (vedi discussione nota (5)).</li>
</ol>4. analisi di sequenziamento dati e generazione di replica profili
<ol><li>Allineare sequenze di genoma di Saccharomyces cerevisiae sacCer3 utilizzando Gsnap12 (Vedi nota di discussione (6)).</li>
	<li>Determinare a base pair leggi di profondità utilizzando "genomeCoverageBed" software13 degli Bedtools.</li>
	<li>Se presente, rimuovere punte artefactual nelle profondità di lettura, (vedi discussione nota (7)).</li>
	<li>Liscio leggere profondità dal cromosoma utilizzando mediane in un 20kb scorrevoli finestra utilizzando la funzione "runMean" in R pacchetto "caTools" (vedi discussione nota (8)).</li>
	<li>Trama leggere profondità in fase S, G2 presto e tardi G2 come percentuale della profondità di lettura completamente replicata (Vedi nota di discussione (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>

Gli autori non hanno nulla a rivelare.

Mentre questa tecnica è robusta e relativamente dritto in avanti, particolare attenzione deve essere rivolta al seguente:
(1) si consiglia che culture crescono per almeno 12 ore prima che raggiungano la fase di log, poiché le differenze si manifestano nel ciclo cellulare distribuzioni se culture vengono vendemmiate, dopo aver raggiunto la densità desiderata solo 4 h dopo l'inoculazione. La nostra ipotesi sono che una distribuzione del ciclo cellulare che ha raggiunto un equilibrio relativam...

In più siti discreti, chiamati origini di replicazione, da cui la replica forcelle procedere in entrambe le direzioni (rivisto in Fragkos et al., 20151) viene avviata la replica del genoma eucariotico. Origini variano nella loro tempistica e l'efficienza di cottura, e sono state sviluppate diverse tecniche per monitorare l'attività di origine di replica e delucidare le cause di questa variazione. L'attività delle singole origini può essere dedotto dai livelli di single-stranded DNA2, che si forma intorno origini attivi, o utilizzando 2D elettroforesi del gel per monitorare specifiche replica intermedi3, entrambi i quali possono essere rilevati con sonde radioattive. Entrambe queste tecniche sono più facilmente applicate in S. cerevisiae rispetto in cellule di mammifero, perché origini sono limitati a specifiche sequenze conosciute nel precedente. Con l'avvento dei microarray, è diventato possibile valutare origine sparando a livello globale. Questo è stato fatto prima di etichettatura del DNA con gli isotopi pesanti, rilasciando le cellule da un blocco di G1 in medi contenenti isotopi leggeri e monitoraggio quindi la formazione di pesante-leggero ibrido DNA attraverso il genoma4. L'introduzione di sequenziamento ad alta permesso simili genoma monitoraggio dell'attività di origine senza l'obbligo di etichettatura isotopica costosi. Le cellule sono state ordinate in un citometro a flusso in base al contenuto di DNA e il loro DNA sottoposti a sequenziamento profondo. Perché copertura sequenza procede da 1x a 2x nel corso della fase S, tempo di replicazione relativa può essere valutata confrontando la lettura profondità delle cellule in fase S a quelli in G1 o G25,6. Queste tecniche, in particolare applicate ai lieviti, ha portato ad una più profonda comprensione di come sequenza di DNA, struttura della cromatina e DNA replica proteine regolano la efficienza e la tempistica di origine.
L'inizio non solo successo della replicazione del DNA, che si svolge alle origini, ma anche il completamento della replica, che si verifica dove si incontrano le forcelle replica richiede fedele trasmissione dell'informazione genetica durante la proliferazione cellulare. Come l'inizio della replica, i tempi di completamento della replica variano in tutto il genoma con alcune regioni restanti non replicate anche tardi nel ciclo cellulare. Tali regioni possono essere particolarmente distante dalle origini di replicazione attiva o possono contenere sequenze o strutture di cromatina che ostacolano la polimerasi del DNA. Replica ritardata regioni possa manifestarsi come siti fragili, che sono associate con rottura cromosomica e più elevati tassi di mutazione e sono stati implicati nel cancro e invecchiamento7,8,9. Tuttavia, nonostante l'importanza del corretto completamento della replica del DNA nel mantenimento della stabilità del genoma, la nostra comprensione di come e dove questo avviene è rimasta lontano dietro quella dell'iniziazione di replica. E mentre singoli geni cui replica tarda è stata associata con la malattia sono stati studiati con, ad esempio, qPCR10globali studi rivolti a chiarire le posizioni e cause di ritardo replica sono mancati. Qui descriviamo una tecnica che ci si riferisce a come "G2 seq" in cui uniamo la citometria a flusso con sequenziamento ad alta per far luce sul completamento della replicazione del genoma in lievito11. Con modifiche minori, il presente protocollo può essere adattato a tutte le celle che possono essere ordinato flusso in base al contenuto di DNA.

<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

Numerose tecniche sono state sviluppate per seguire l'avanzamento della replica del DNA attraverso la fase S del ciclo cellulare. La maggior parte di queste tecniche sono state dirette verso delucidazione della posizione e la tempistica di apertura di duplicazione del genoma piuttosto che il suo completamento. Tuttavia, è fondamentale che capiamo regioni del genoma che sono ultima per completare la replica, perché queste regioni soffrono i livelli elevati di rottura cromosomica e mutazione, e sono stati associati con la malattia e invecchiamento. Qui descriviamo come siamo ad una tecnica che è stata utilizzata per monitorare l'inizio di replica per invece identificare quelle regioni del genoma Ultima replica completa. Questo approccio si basa su una combinazione di citometria a flusso e sequenziamento ad alta. Anche se la relazione si concentra sull'applicazione di questa tecnica di lievito, l'approccio può essere utilizzato con tutte le celle che possono essere ordinate in un citometro a flusso in base al contenuto di DNA.

Abbiamo utilizzato la procedura descritta sopra per identificare siti tardi replicanti in lievito. Questo approccio utilizzando una regione conosciuta di replicante tardiva su un cromosoma artificiale di test hanno dimostrato che la tecnica per essere precisi e affidabili. I nostri risultati hanno dimostrato anche l'importanza biologica di tempestivo completamento della replica mostrando che una tardiva replicante regione sul cromosoma 7, che abbiamo identificato come fine la replica in b...

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: Un'elevato Throughput sequenziamento-base tecnica per identificare replica ritardata regioni del genoma

Descriviamo una tecnica per combinare la citometria a flusso e sequenziamento ad alta per identificare regioni tarda replicazione del genoma.

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 Views.  Fred Hutchinson Cancer Research Center. Descriviamo una tecnica per combinare la citometria a flusso e sequenziamento ad alta per identificare regioni tarda replicazione del genoma.

Video: 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.

Una piattaforma robotica per high-throughput Protoplast Isolamento e Trasformazione

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 ...

Ricerca

Genetica

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.

Mappatura delle interazioni RNA-RNA globalmente utilizzando Psoralen biotinilato

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.

Scansione retrovirale: Mappatura dei siti di integrazione MLV per definire regioni regolatori specifiche per le cellule

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.

Generazione di immunoprecipitazione della cromatina nativo sequenziamento librerie per analisi di densità del nucleosoma

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.

Isolamento del DNA robusto e costruzione della libreria High-throughput sequenziamento per campioni di erbario

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.

Microinjection di embrione e tecnica di trapianto per la manipolazione del genoma di Nasonia vitripennis

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.

Identificazione di High-throughput di combinazioni di farmaci sinergici con il metodo2 di sovrapposizione

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.

Promotore cattura Ciao-c: Ad alta risoluzione, Genome-wide profilatura delle interazioni del promotore

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.

Identificazione di High-throughput di sequenze regolatorie del Gene mediante sequenziamento di nuova generazione di cromosoma circolare conformazione catturare (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-mediata Editing genomico agente patogeno fungoso umano Candida albicans

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