This work was supported by grant NIH GM117446 to A.B.

1. Preparation of Cells for Flow Cytometry Sorting
<ol>
	<li>Inoculate 15 mL test tubes containing 8 mL each of YEPD broth such that the cultures reach a density of 5 x 106 to 1.5 x 107 cells per mL after overnight growth (see discussion note 1).</li>
	<li>Spin down yeast cells (1,400 x g, at room temperature or 4 &#176;C) in a 15 mL screw cap centrifuge tube for 5 min, resuspend cells in 1.5 mL 70% ethanol, and transfer to a 1.6 mL microfuge tube, letting this sit for 1 h at room temperature or a...

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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. 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While this technique is robust and relatively straight forward, particular attention should be devoted to the following:
(1) We recommend that cultures grow for at least 12 h before they reach log phase, since differences manifest in cell cycle distributions if cultures are harvested after having reached the desired density just 4 h after inoculation. Our assumption is that a cell cycle distribution that has reached a relatively stable equilibrium better represents a &#34;real&#34; log phase d...

Eukaryotic genome replication is initiated at multiple discrete sites, called origins of replication, from which replication forks proceed in both directions (reviewed in Fragkos et al., 20151). Origins vary in both their timing and efficiency of firing, and several techniques have been developed to monitor replication origin activity and elucidate the causes of this variation. The activity of individual origins can be inferred from levels of single-stranded DNA2, which forms around active origins, or by using 2D gel electrophoresis to monitor specific replication intermediates3, both of which can be detected with radioactive probes. Both of these techniques are more easily applied in S. cerevisiae than in mammalian cells, because origins are limited to specific known sequences in the former. With the advent of microarrays, it became possible to assess origin firing globally. This was first done by labeling DNA with heavy isotopes, releasing cells from a G1 block into medium containing light isotopes, and then monitoring the formation of heavy-light hybrid DNA across the genome4. The introduction of high throughput sequencing allowed similar genome-wide monitoring of origin activity without the requirement of expensive isotopic labeling. Cells were sorted in a flow cytometer according to DNA content and their DNA subjected to deep sequencing. Because sequence coverage proceeds from 1x to 2x over the course of S phase, relative replication timing can be assessed by comparing read depths of cells in S phase to those in G1 or G25,6. These techniques, particularly applied to yeast, led to a deeper understanding of how DNA sequence, chromatin structure, and DNA replication proteins regulate origin timing and efficiency.
Faithful transmission of genetic information during cellular proliferation requires not only successful initiation of DNA replication, which takes place at origins, but also successful completion of replication, which occurs where replication forks meet. Like initiation of replication, the timing of completion of replication varies across the genome with certain regions remaining unreplicated even late in the cell cycle. Such regions may be particularly distant from active replication origins or may contain sequences or chromatin structures that impede DNA polymerases. Late replicating regions can manifest themselves as fragile sites, which are associated with chromosomal breakage and higher mutation rates, and have been implicated in cancer and aging7,8,9. However, despite the importance of proper completion of DNA replication in maintenance of genome stability, our understanding of where and how this takes place has lagged far behind that of replication initiation. And while individual genes whose late replication has been associated with disease have been studied with, for example, qPCR10, global studies directed at elucidating the locations and underlying causes of late replication have been lacking. Here we describe a technique we refer to as &#34;G2 seq&#34; in which we combine flow cytometry with high throughput sequencing to shed light on the completion of genome replication in yeast11. With minor changes, this protocol can be adapted to any cells that can be flow-sorted according to DNA content.

<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

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 been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We have used the procedure described above to identify late replicating sites in budding yeast. Testing this approach using a known late replicating region on an artificial chromosome proved the technique to be accurate and reliable. Our results have also demonstrated the biological importance of timely completion of replication by showing that a late replicating region on chromosome 7, which we identified as late replicating on the basis of our G2-seq results, was lost approximately thre...

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

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

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 ...

The overall goal of this procedure for deep sequencing DNA from cells that have been flow sorted according to cell cycle phase is to identify regions of the genome that replicate extremely late in the cell cycle. This method can help answer key questions in the DNA replication field such as which regions of the genome have trouble completing DNA replication and are therefore particularly vulnerable to genome rearrangements. The main advantage of this technique is that even though it is very simple from an experimental standpoint, it provides a surprisingly rich view into the dynamics of genomic replication.To begin, inoculate 15 milliliter test tubes containing eight milliliters of YEPD with yeast cells of interest. Synthetic or rich medium is acceptable. Culture the cells overnight for at least 12 hours to collect them during their real log phase distribution.The following day when the cultures are at a density of five to 15 million cells per milliliter, spin down the cells and resuspend them in 1.5 milliliters of 70%ethanol. Then transfer the suspension to a 1.6 milliliter microfuge tube and let the cells incubate at room temperature for an hour or for at least three hours on ice. After the inoculation, spin down the cells and resuspend them in one milliliter of sodium citrate.Finally, briefly sonicate the cells twice. Then repeat the centrifugation cycle and resuspend the yeast in one milliliter of sodium citrate containing RNase. Allow the RNase to react with the yeast at 50 degrees Celsius for an hour or overnight at 37 degrees Celsius in a heat block or a water bath.After the RNase treatment, add 50 microliters of proteinase K solution to the mixture and continue the reaction for an hour at 50 degrees Celsius. Then spin down the cells and resuspend the cells in one milliliter of sodium citrate. Next, centrifuge the cells and aspirate the supernatant using a vacuum.Then in dim light resuspend the cells in one milliliter of sodium citrate with green nucleic acid stain. Now incubate in the yeast in the dark for an hour at room temperature. To proceed, count the cells using a cell sorter using 530 nanometer emission data.Sort them according to their DNA content into their cell cycle phases, G1 S, early G2, and late G2.Collect at least 1.6 million haploid cells from each phase. Promptly after sorting the cells, spin them down for 20 minutes. Aspirate the supernatant and freeze the pellet at negative 20 degrees Celsius for storage.We have found that the single most critical step for this procedure is simply spinning the cells down promptly after collecting them, an hour the most after sorting. This extraction is based on a yeast genomic DNA extraction kit. Begin with adding 120 microliters of digestion buffer and five microliters of yeast lytic enzyme.Then blend the cells into the reaction mixture using a vortex and incubate the mixture at 37 degrees Celsius for 40 to 60 minutes. Next, add 120 microliters of lysis buffer and vortex the mixture at high speed for 10 to 20 seconds. Then add 250 microliters of chloroform and for one minute thoroughly mix the tube contents.Next, spin down the tube at maximum speed for two minutes. Then transfer the supernatant onto the DNA-binding column. Spin the supernatant through the column using a one-minute maximum speed centrifugation cycle.To wash the DNA on the column membrane, transfer the column to a new collection tube and apply 300 microliters of DNA washing buffer. Then repeat the centrifugation at maximum speed for one minute. Repeat this wash step one more time.To collect the DNA, transfer the spin column to a fresh tube. Add 60 microliters of water and wait one to three minutes. Then centrifuge the column at maximum speed for 10 seconds to isolate the water containing the eluted DNA.Now add between five and 195 microliters of double-stranded DNA high-sensitivity buffer containing reagent at a one to 200 concentration. Then measure the sample's fluorescence to calculate the yield. Expect to collect between 10 and 50 nanograms of DNA.Now use sonication to break up the DNA into fragments that are between 250 and 350 base pairs. Then use a standard kit to prepare a DNA library and sequence it using at least 10 million 50 base pairs single-end reads. The described procedure was used to identify late replicating sites in budding yeast.Normal cells were compared with those lacking Sir2, a deacetylase protein. The raw data contained large spikes mostly due to the presence of TY elements. These spikes were removed by capping the read depths to 2.5 times the median red depth for each sample.The de-spiked data was then smoothed with a 20 kb sliding window. Finally, the data was normalized and plotted as ratios to levels in G1.A completely unreplicated cell would appear at 0.5 and a completely replicated cell would appear at one. In this data from chromosome seven, there was evidence of a known late replicating region in the Sir2 mutant.After watching this video, you should have a good understanding of how to identify those regions of the genome that are last to complete DNA replication and that are therefore particularly vulnerable to DNA breaks and other problems that are associated with late replication.

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Research

JoVE Journal

Genetics

5.7K Views.  Fred Hutchinson Cancer Research Center. The overall goal of this procedure for deep sequencing DNA from cells that have been flow sorted according to cell cycle phase is to identify regions of the genome that replicate extremely late in the cell cycle. This method can help answer key questions in the DNA replication field such as which regions of the genome have trouble completing DNA replication and are therefore particularly vulnerable to genome rearrangements. The main advantage of this technique is that even though it is very s...