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