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.
