Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Note: ToR can be measured only on growing, unsynchronized cells. The procedure should begin with at least 1 – 2 x 10⁶ fast growing cells, which will usually result in ~1 x 10⁵ cells in S phase (the rate limiting step). It is recommended to conduct each experiment using two or three replicates. The entire process of CNR-ToR can be completed within one week — two days should be dedicated to all steps up to library preparation, one to two days are needed for sequencing and an additional day is necessary for the initial data analysis.

1. Collection of Cells from Culture

NOTE: The protocol is written for cells growing in culture in 10 cm plates (containing approximately 2 – 5 x 10⁶ cells), but can be easily adjusted to other platforms.

1. For cells that were grown in suspension, proceed to fixation (section 2).
2. For adherent cells, aspirate and wash the plate with 3 mL PBS without Ca²⁺ and Mg²⁺.
3. Discard the PBS and incubate the cells for 5 min at 37 °C with 1 mL commercial trypsin-EDTA until cells detach.
   NOTE: The duration of the trypsin treatment should be adjusted to each cell type.
4. Add 3 mL culture media to neutralize the trypsin and collect cells in a 15 mL conical tube or a 5 mL polystyrene tube. Keep on ice.

2. Fixation

NOTE: For this part, all steps should be done at 4 °C.

1. Centrifuge the cells at 300 x g for 5 min at 4 °C.
2. Aspirate and wash cells twice with 1 mL cold PBS.
3. Resuspend cells in 250 µL (total) cold PBS.
4. While gently vortexing the tube, slowly add dropwise 800 µL of -20 °C 100% ethanol. This leads to a final ethanol concentration of 70 – 80%.
   NOTE: High purity ethanol is recommended at this step.
5. Incubate cells on ice for 30 min.
   NOTE: At this stage cells can be kept for a few days at 4 °C or for a few months at -20 °C.

3. PI Staining

1. Centrifuge the cells at 500 x g for 10 min at 4 °C.
2. Aspirate the supernatant carefully and wash cells twice with 1 mL cold PBS.
3. Aspirate and resuspend each sample with the following mixture: 1 mL PBS, 5 µL 10 mg/mL RNaseA, 50 µL 1 mg/mL propidium iodide (PI; mix bottle before use). Adjust concentration up to ~2 x 10⁶ cells/mL).
   NOTE: Keep out of light — PI is light sensitive.
4. Filter through 35 µm mesh to a 5 mL polystyrene tube, and close with Parafilm.
5. Incubate at RT in the dark, for 15 – 30 min.
   NOTE: Cells are now ready for FACS analysis. If necessary, the stained cells can be stored for at least 24 h at 4 °C in the dark.

4. Sort

1. Sort cells using a FACS machine. Use the 561 nm laser to differentiate cells based on their PI intensity. Other lasers near the 535 nm excitation maximum like 488 nm or 532 can be used depending on the FACS machine configuration.
2. For optimal results, use the smallest nozzle recommended for the specific cell size (for most cells 85 µm). Prioritize purity modes over yield. Use a steady slow flow, usually up to 300-500 events/s, with a sheath pressure of 45 psi.
3. Using gating, discriminate dead cells and subcellular debris (low FCS and high SSC) by Plotting FCS vs SSC. From the viable cells discriminate doublets by plotting SSC-Width (W) vs SSC-Height (H) followed by a FSC-W vs FSC-H plot and by PI- W vs PI-H (doublets will have the same H-value but bigger W-value). For the viable single cells draw a histogram of the PI-Area (A) intensity which represents the DNA content of the cells.
4. Sort cells into G1 and S phases, as shown in Figure 1. 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.

Figure 1. Cell cycle phase determination based on PI intensity. Histogram showing the distribution of the cellular DNA content (measured by PI-Area) of mouse embryonic fibroblast (MEF) population. The DNA content is used to sort the population into two sub-populations i) G1 cells (2N DNA content) and ii) S phase cells (2N – 4N DNA content), using the marked regions. Please click here to view a larger version of this figure.

NOTE: The purpose of the collection of G1 cells is to account for biases in the sequencing efficiency between different genomic regions. An alternative approach is to use G1 arrested cells from the same cell type. This approach gives cleaner results (since it minimizes S phase contamination) but it may introduce biases stemming from genetic differences between the arrested cells and the measured cells.

5. Sort in cold conditions and collect sorted cells into 1.5/2 mL tubes. Keep tubes on ice following the sort.
   NOTE: In order to improve DNA recovery, it is better to use low binding tubes or to use tubes coated with 4 – 5% BSA for 1 – 3 h at 4 °C ¹⁸.

5. DNA Purification

1. For each sample (G1 and S) purify DNA using a DNA purification kit.
   NOTE: When using the commercial kit, elute with 400 µL elution buffer into a 2 mL tube for high yield, as recommended by the manufacturer.
2. Check DNA concentration using fluorometer.
   NOTE: From 100,000 mammalian cells collected by the FACS, one should get ~ 1 µg of DNA. At this stage DNA can be kept for a few days at 4 °C or at -20 °C for long storage.

6. Sonication

1. Transfer DNA into a 1.7 mL tube that is compatible with the magnetic stand used.
2. Concentrate DNA using 2x SPRI beads according to the manufacturer's instructions, using a magnetic stand, and elute in 50 µL elution buffer.
3. Shear DNA with a focused ultrasonicator to an average target peak size of 250 bp. Use the following settings for a 50 µL DNA sample: 50 W, 20% Duty factor, 200 Cycles per burst, 20 °C, 120 s.
4. Verify the size of the sheared DNA by electrophoresis. The recommended size distribution is 200 – 700 bp with a peak at ~ 250 bp.
   NOTE: At this stage DNA can be kept for a few days at 4 °C or at -20 °C for long storage.

7. Library Preparation, and Sequencing

NOTE: Many library preparation kits and different sequencing platforms should work similarly to the ones used by us and mentioned in the materials section. Actually in the past, ToR maps were generated using a very similar method with microarray platforms².

1. Prepare libraries using any commercial library preparation kit.
2. At the end of library preparation, select size using magnetic beads for 300 – 800 bp.
3. After preparing libraries, measure DNA concentration using fluorometer.
4. Measure DNA size using electrophoresis.
5. Perform sequencing on any platform.
   NOTE: Sequencing at least 10 M reads per sample is recommended. This depth is equivalent to a read approximately every 300 bp (for ~3 Gb size genomes), and is sufficient for ToR measurements at a resolution of 50 – 100 kb. Increasing the depth will result in a decrease in the size of the windows and thus will enable increasing resolution with higher certainty. Paired end sequencing is not necessary in this protocol since only coverage information is collected. It, however, may help in resolving the location of reads that contain repetitive sequences.

8. Analysis

NOTE: Data analysis is based on the method used by A. Koren et al.¹⁹.

1. Map sequencing data to the corresponding genome using bowtie2 or any reliable short read aligner. Define varying-size, equal-coverage chromosomal windows as segments covered by 200 reads in the G1 fraction and count S phase reads in the same windows.
2. Calculate the S/G1 ratio for each window. This should generate a map with large fluctuations in the S/G1 ratio along the genome (Figure 3). A good control for the reliability of the ToR measurements is to compare this map to the G1/G1 ratio (from two separate measurements of G1) which should be much flatter.
3. Normalize data to 0 mean and 1 SD by subtracting from each value the average value of all windows (excluding the X chromosome) and dividing the results by the standard deviation of the S/G1 of all windows. This is done in order to convert to z scores and enable comparison between different experiments.
4. Remove all gap regions listed by the UCSC genome browser as well as each remaining inter gap fragment containing fewer than 15 data windows.
5. Smooth the remaining fragments with a cubic smoothing spline via the Matlab function csaps with a parameter of 10⁻¹⁶ and interpolate at set points every 100 kb.
   NOTE: Parameters of smoothing and interpolation should be adjusted based on the depth of the data. Other suitable smoothing methods and functions exist and can be used.
6. After visually confirming the reliability of each replicate, merge all the reads and calculate a deeper resolution profile by performing the same process described above on this data.