Protocol

Genome-Wide Analysis of DNA Synthesis by BrdU Immunoprecipitation on Tiling Microarrays (BrdU-IP-chip) in Saccharomyces cerevisiae

Molecular and Computational Biology Program, University of Southern California, Los Angeles, CA 90089, USA

¹Corresponding author (oaparici{at}usc.edu).

INTRODUCTION

The incorporation of thymidine analogs, such as 5-bromo-2'-deoxyuridine (BrdU), into newly synthesized DNA is a powerful tool for analysis of DNA replication, repair, and other aspects of DNA metabolism. In Saccharomyces cerevisiae, several assays have been developed to identify chromosomal DNA that has incorporated BrdU. Here we describe an approach that couples BrdU immunoprecipitation with DNA microarrays (BrdU-IP-chip) supplied by Roche NimbleGen to enable the genome-wide identification of BrdU-labeled chromosomal DNA. In this procedure, yeast cells that have been engineered to assimilate BrdU from the growth medium and incorporate it into replicating DNA are grown in the presence of BrdU under experimental conditions. These cells are harvested, the genomic DNA is isolated and randomly sheared, and the BrdU-labeled DNA is then immunoprecipitated. This immunoprecipitated DNA is polymerase chain reaction (PCR)-amplified, labeled with a fluorophore, and cohybridized along with a reference sample (typically total genomic DNA not subject to immunoprecipitation, but amplified and labeled with a different fluorophore) onto DNA microarrays. The data are then normalized, and chromosomal regions of BrdU incorporation (BrdU peaks) are identified. BrdU peak heights can be quantified and compared with BrdU peak heights at different genomic locations or under different experimental conditions (e.g., different mutants or time points). BrdU-IP-chip has many potential applications and has already been used to identify replication origins, make quantitative comparisons of origin firing between strains, and examine replication fork progression.

MATERIALS

Reagents

Use Milli-Q water to prepare all stock solutions.

Anti-BrdU antibody (GE Healthcare)

Array Reuse Kit (NimbleGen) (optional; see Step 57)

Blocking DNA (50X)

BrdU (100X)

Dual-Color DNA Labeling Kit (includes Cy5 and Cy3 random 9-mers, exo-Klenow; NimbleGen 05223547001)

Dual-color DNA labeling kits from other vendors may also be used.

EDTA (10 mM)

Elution buffer for genomic DNA

Ethanol

GenomePlex Complete Whole Genome Amplification (WGA) Kit (Sigma WGA2)

Similar amplification kits from other suppliers may be suitable but have not been tested.

Hybridization Kit (includes 2X hybridization buffer, hybridization component A, alignment oligo; NimbleGen 05223474001)

Immunoprecipitation (IP) buffer for genomic DNA, prechilled on ice

Lysis buffer for genomic DNA

MinElute PCR Purification Kit (includes spin columns, PE and EB buffers, and collection tubes; QIAGEN 28004)

NaCl (5 M)

NimbleGen 385K microarrays for Saccharomyces cerevisiae (e.g., whole genome tiling array; B2436001-00-01)

Other microarray formats are available but may require different coverslips and sample volumes; multiplex array formats require additional equipment and supplies available from NimbleGen.

PBS for genomic DNA (10X)

PCR reagents and primers for analysis of genomic DNA sites known to incorporate and not incorporate BrdU (see Step 34)

Phenol:chloroform:isoamyl alcohol (25:24:1; PCI)

Store at 4°C.

Protein G-Sepharose beads (50% slurry in PBS for genomic DNA)

Sodium azide can be added as a preservative to the slurry at 0.01% (w/v). This protocol works well with magnetic protein G beads (Dynabeads Protein G; Invitrogen) (see "Immunoprecipitation of BrdU-Labeled DNA").

QIAquick PCR Purification Kit (QIAGEN 28104) or similar product

Reagents for agarose gel electrophoresis (use if determining DNA shear size; see Step 15)

Reference DNA or yeast cells for preparing reference DNA

The reference DNA sample will be fluorescently labeled and cohybridized onto the microarray with the immunoprecipitated (IP) DNA (see "Incorporation of BrdU and Harvesting of Yeast Cells").

RNase A (DNase-free, 20 mg/mL; Sigma)

Store at -20°C.

S. cerevisiae cells

The analysis described in this procedure must be conducted on yeast cells that have been engineered to assimilate BrdU from the growth medium and incorporate it into DNA. This can be accomplished in yeast by overexpressing herpes simplex virus thymidine kinase to phosphorylate nucleosides (Lengronne et al. 2001) and is made more efficient with the combined expression of an equilibrative nucleoside transporter to facilitate efficient uptake of extracellular nucleosides (or their analogs) (Vernis et al. 2003; Viggiani and Aparicio 2006).

Sodium azide

SSC (3X), prechilled to 4ºC

TE (pH 7.6)

Tris-buffered saline (TBS) for yeast, prechilled on ice

Wash Buffer Kit (NimbleGen 05223504001)

Yeast extract-peptone-dextrose growth medium (YEPD) or other medium

Equipment

Agarose gel (2%) and equipment for electrophoresis (use if determining DNA shear size; see Step 15)

Array Processing Accessories Kit (NimbleGen 05223539001)

Bunsen burner

Centrifuge (tabletop, refrigerated, with swinging-bucket rotor), prechilled to 4ºC

Compressed air

Dry ice/ethanol bath (optional; see Step 7)

Alternatively, liquid nitrogen can be used to freeze the cells.

Equipment for culturing yeast cells

FastPrep FP120 cell disrupter (MP Biomedicals)

Standard vortexers may also be used.

Glass beads, 425-600 µm in diameter (Sigma G8772), washed and heat-sterilized

Heat blocks preset to 42ºC (optional; see Step 47), 55ºC, 65ºC, 95ºC

Hypodermic needles (26-gauge x 1 in.), with small syringe for holding

Ice

Ice-water bath

Incubator or water bath preset to 37ºC

Incubator with shaker preset to 30ºC

LifterSlip coverslips of the appropriate dimensions to cover array features completely (Thermo Scientific)

This protocol describes the hybridization using 22 x 22 mm LifterSlips with a capacity of ~40 µL.

Microarray analysis software (e.g., Genepix, ImaGene, NimbleScan)

Microarray hybridization chambers (Corning)

Microarray scanner (Axon GenePix; MDS Analytical Technologies)

Microcentrifuge

Micropipettor and tips (standard and wide-bore)

Minicentrifuge for glass slides

NanoDrop spectrophotometer (Thermo Scientific)

Similar devices that conserve the DNA sample may also be used.

Ozone-free equipment enclosure (e.g., a room or box with an ozone catalyst system)

Pipettes (serological) and pipetting device

Refrigerator preset to 4ºC or freezer preset to –20ºC

Slide box

Sonicator (Branson 250 with microtip attachment)

Other sonicators may also be used.

Spectrophotometer (visible light to measure yeast cell concentration)

Thermocycler

Tube rotator

Store in cold room or refrigerator.

Tubes, 15- or 50-mL conical, screw-cap

Tubes, 5-mL polypropylene, snap-cap

Tubes, microcentrifuge, 1.5-mL

Tubes, microcentrifuge, 2-mL with gasket-sealed screw-caps, prechilled (required for FastPrep cell disrupter) (e.g., VWR)

Vacuum concentrator

Vacuum grease

Waterbath or hybridization oven in dark preset to 42ºC

METHOD

Incorporation of BrdU and Harvesting of Yeast Cells

Because experimental designs may vary substantially (see Szyjka et. al. [2008]; Knott et al. [2009a]), we describe only the addition of BrdU to the growth medium and cell harvesting. For the reference DNA sample (to be fluorescently labeled and cohybridized onto the microarray with the IP DNA), we typically use genomic DNA prepared from G1-arrested cells (not labeled with BrdU). However, BrdU-labeled DNA may be used, and DNA from different cell cycle stages or time points may be used. For the former, genomic DNA from 10-20 mL of G1-arrested cells (OD₆₀₀~ 0.5-1.0) should be prepared (Steps 3-22) in parallel with BrdU-incorporated genomic DNA. For the latter, 1 ug of BrdU-incorporated genomic DNA obtained at Step 20 is used. In both cases, reference DNA should be amplified to account for potential differences in amplification efficiencies of different sequences.

1. Grow 10-20 mL of S. cerevisiae cells to an OD₆₀₀ ~ 0.5-1.0 in YEPD (or other) medium.

2. Add BrdU to a final concentration of 400 µg/mL and incubate for the desired period. For very short incubations with BrdU (e.g., 10-15 min), doubling the concentration of BrdU may be helpful.

3. To harvest, add sodium azide to 0.1% final concentration and transfer the culture to a 15- or 50-mL conical screw-cap tube.

4. Pellet the cells by centrifugation in a swinging-bucket rotor at 1500g for 5 min at 4°C. Discard the supernatant.

5. Resuspend the cell pellet by pipetting up and down in 20 mL of ice-cold TBS for yeast. Pellet the cells by centrifugation at 1500g for 5 min at 4°C.

6. Resuspend the cell pellet in 1 mL of ice-cold TBS for yeast with a micropipettor and transfer to a chilled, 2-mL microcentrifuge tube with a gasket-sealed screw-cap.

7. Pellet the cells in a microcentrifuge at full speed (~16,000g) for a few seconds. Remove the supernatant without disturbing the cell pellet.
At this point, the cell pellet may be frozen rapidly using a dry ice-ethanol or liquid nitrogen bath and stored at –20°C.

Cell Lysis and Preparation of Genomic DNA

8. Thaw the cell pellet (if necessary) and resuspend the cells in 500 µL of lysis buffer for genomic DNA.

9. Add an equal volume (~0.6 mL) of glass beads to the cell suspension.

10. Tightly cap the tube and place it into a FastPrep instrument. Run the FastPrep at power setting 5.5 for 45 sec.
Other vortexers may be used, but specific settings to achieve maximal cell breakage will vary.

11. Remove the tube from the FastPrep and centrifuge at full speed for a few seconds to collapse any foam. Add 25 µL of 5 M NaCl to each sample.

12. Repeat Step 10.

13. Process the tube as follows:

i. Invert the tube and flick to knock the beads and solution away from the bottom of the tube.

ii. Puncture the bottom of the tube twice with a red-hot 26-gauge needle (attached to a small syringe to hold the needle safely).

iii. Immediately insert the tube into a 5-mL polypropylene snap-cap tube with its cap removed (it may only fit partially).

iv. Centrifuge in a swinging-bucket rotor at 350g for 2 min to collect the lysate.

v. Remove the tube combination from the centrifuge.

vi. Remove the FastPrep tube, which should contain only the beads, from the 5-mL tube, which contains the cell lysate.
The lysate is partially pelleted at this point.

14. Decant the soluble lysate to a fresh microcentrifuge tube and leave behind the pellet, which is mainly cell debris (some carryover is acceptable).

15. Sonicate as follows:
i. Insert the microtip horn of a sonicator about halfway down the depth of the lysate in the microcentrifuge tube.
ii. Sonicate for 20 sec with constant output on low power. With the Branson 250 sonicator (with microtip attachment), use power setting 2.0 and 100% duty cycle.
iii. Chill the sample on ice between rounds of sonication.
Specific settings must be determined if a different sonicator is used. The DNA shear size is an important parameter that should be determined for a particular instrument. Determine the DNA shear size by analyzing the DNA by 2% agarose gel electrophoresis; it should appear as a smear with the majority of DNA in the 200- to 1000-bp range.
See Troubleshooting.

16. Repeat Step 15 (multiple times if smaller DNA shear size is desired).

17. Extract the sample as follows:

i. Add 500 µL of PCI to each sample and vortex vigorously.

ii. Separate the phases by centrifugation at full speed (~16,000g) for 5 min.

iii. Transfer the aqueous phase (~450 µL) to a new microcentrifuge tube.

18. Repeat Step 17.

19. Add 1 mL of ethanol to each sample, mix, and centrifuge at full speed (~16,000g) for 15 min. Decant the supernatant and allow residual liquid to evaporate.

20. Resuspend the pellet in 100 µL of TE (pH 7.6) + 1 µL of RNaseA and incubate for 30 min at 37°C.

21. Purify the samples using a QIAquick PCR Purification Kit according to the manufacturer’s instructions. Elute with 50 µL of TE (pH 7.6).

22. Measure the DNA concentration using a NanoDrop spectrophotometer or similar device.
At least 2.5 µg (as measured with NanoDrop) of genomic DNA should be recovered from a 20-mL cell culture with OD ~ 1. Samples may be stored overnight at 4°C or for a few weeks at -20°C.

Immunoprecipitation of BrdU-Labeled DNA

In these steps, BrdU-labeled genomic DNA that has been isolated and sheared is denatured and immunoprecipitated using an antibody specific to BrdU. Reference DNA is not subjected to this procedure and is set aside until the amplification in Steps 35-38. If immunoprecipitated DNA is to be analyzed with high-throughput sequencing, we do not recommend a blocking step (see below), because it uses nonhomologous DNA that may remain in the immunoprecipitated sample and complicate sequencing analysis. Instead, we suggest the use of Dynabeads Protein G, which does not require blocking.

23. Combine in a microcentrifuge tube:

Sheared, genomic, BrdU-labeled DNA (from Step 22) 1 µg Blocking DNA (50X; 5 µg/µL) 2 µL (10 µg) PBS for genomic DNA (10X) 5 µL H2O to 50 µL

If different amounts of DNA are used, alter the amounts and volumes appropriately, maintaining all concentrations.

24. Process the sample as follows:

i. Heat the sample on a heat block for 10 min at 95°C to denature the DNA.

ii. Snap-cool the sample in an ice-water bath for a few minutes.

iii. Briefly centrifuge the tube in a microcentrifuge to collect the solution in the bottom of the tube.

25. Incubate with anti-BrdU antibody:

i. Dilute the appropriate amount (e.g., 1:1000) of anti-BrdU antibody in ice-cold IP buffer for genomic DNA.

ii. Add 200 µL of diluted antibody to the denatured DNA sample.

iii. Incubate with gentle agitation (e.g., using a tube rotator) for 1 h (up to overnight) at 4°C.

26. Briefly centrifuge to collect the sample in the bottom of the tube. Using a wide-bore pipette tip, add 30 µL of a 50% suspension of Protein G-Sepharose beads in PBS for genomic DNA and incubate on a tube rotator for 1 h at 4°C.

27. Gently pellet the beads using a microcentrifuge at ~800g for 1 min at room temperature. Carefully remove the supernatant, avoiding the beads.

28. Add 1 mL of ice-cold IP buffer for genomic DNA and place the sample on a tube rotator for ~3-5 min at room temperature. Repeat Step 27.

29. Repeat Step 28 two additional times.

30. Add 1 mL of TE (pH 7.6) and place on a tube rotator for ~3-5 min at room temperature. Repeat Step 27.

31. Add 100 µL of elution buffer for genomic DNA to the beads and incubate in a heat block for 15 min at 65°C.

32. Pellet the beads as in Step 27. Transfer the eluate to a fresh microcentrifuge tube.

33. Purify the eluate using a MinElute PCR Purification Kit according to the manufacturer’s protocol with the following modifications:

i. Perform two washes with PE Buffer.

ii. Elute with 11 µL of 0.2X EB prewarmed to ~50°C.

34. Store samples overnight at 4°C, or for a few weeks at -20°C. For longer-term storage, dry the DNA samples and place at -20°C or -80°C.
At this point, you may proceed to the DNA amplification. However, before investing time and resources into these steps, it is advisable to confirm the expected level of enrichment (if possible) by direct PCR analysis.
See Troubleshooting.

DNA Amplification

To generate sufficient DNA for analysis, the IP and reference DNA samples must be amplified before labeling the DNA with a fluorophore. Commercially available kits are available for efficiently amplifying sheared DNA fragments.

35. Determine the DNA concentration of the IP and reference samples using a NanoDrop or similar instrument. Approximately 50 ng (as measured by NanoDrop) of IP DNA should be recovered.
See Troubleshooting.

36. Use the GenomePlex Complete WGA Kit to separately amplify 10 ng of IP and 10 ng of reference DNA according to the manufacturer’s protocol.

37. After the amplification reaction, purify the DNA samples using a QIAquick PCR Purification Kit according to the manufacturer’s instructions. Elute using 30 µL of buffer EB.

38. Measure the concentration of each sample with a NanoDrop. More than 3 µg (as measured by NanoDrop) of WGA-amplified DNA should be recovered.
At this point, the samples may be stored at -20°C, or dried for long-term storage.
See Troubleshooting.

Fluorescent Labeling of the DNA

There are various methods for fluorescently labeling DNA samples using fluorophores. This section describes the direct labeling of samples by a Klenow (exo-) extension reaction using Cy-labeled random 9-mers as primers. Always protect Cy dyes and Cy-labeled DNA samples from excessive light exposure.

39. Use the NimbleGen Dual-Color DNA Labeling Kit (or similar) to label the IP DNA with Cy5 and reference DNA with Cy3 according to the manufacturer’s instructions.
The Klenow extension reaction in this step further amplifies the DNA samples. This amplification is dependent on several factors, including the Cy-conjugated random 9-mer primer concentration and the amount of Klenow used. We have found that hybridizations can be performed using less labeled DNA without significantly affecting the data quality; therefore, if desired, in this step both Klenow enzyme and primers can be conserved. However, scaling back on these reagents results in lower amounts of amplified DNA. We have found that a typical reaction to label 1 µg of DNA using primers at OD = 1.0 and 100 U of Klenow produces >20 µg of labeled DNA; using primers at OD = 0.1 and 100 U of Klenow produces ~5 µg of DNA; using primers at OD = 0.1 and 50 U of Klenow produces ~3 µg of DNA.

40. Purify the labeled DNA using a MinElute PCR Purification Kit according to the manufacturer’s protocol. Elute with 10 µL of 10 mM EDTA.
The volume used to dissolve the labeled DNA can be adjusted depending on the anticipated amount of DNA produced in the labeling reaction.

41. Determine the DNA concentration of the Cy-labeled IP and reference samples using a NanoDrop.

42. In a new microcentrifuge tube, for each sample, combine equal amounts of Cy5-labeled IP DNA and Cy3-labeled reference DNA. Dry the combined DNA using a vacuum concentrator.
The amounts required for hybridization may vary. Follow the array manufacturer’s guidelines. We typically combine 1 to 2 µg of IP and reference DNA for hybridization onto NimbleGen yeast arrays; for larger genomes, more DNA should be hybridized.

43. Proceed to the hybridization or store the samples at –20°C, protected from light.

Hybridization to DNA Microarrays

This section describes the hybridization of DNA onto NimbleGen high-density oligonucleotide tiling microarrays representing the yeast genome. Adapted from the protocols supplied by NimbleGen, it uses NimbleGen reagents with standard hybridization chambers and equipment. For other array configurations or platforms, hybridization supplies and protocols included by the microarray manufacturer should be used. For customized microarrays, hybridization temperature and buffers may need to be optimized depending on the array design and slide-surface chemistry.

44. Dissolve the combined IP and reference DNA with 9.5 µL of H₂O.

45. Prepare the hybridization chambers by coating the gaskets with a thin layer of vacuum grease. Clean the inside of each chamber with ethanol and, if desired, compressed air.

46. Using components from a NimbleGen Hybridization Kit, prepare the following hybridization mix for each sample:

Cy-labeled DNA (IP plus reference, from Step 44) 10.9 µL 2X hybridization buffer 19.5 µL Hybridization component A 7.8 µL Alignment oligo 0.8 µL Total volume 39 µL

We describe the hybridization using 22- x 22-mm LifterSlips with a capacity of ~40 µL. For hybridizations requiring different volumes of sample, adjust the volumes above accordingly, maintaining the concentrations.

47. Denature a sample in a heat block for 5 min at 95°C, protected from light.
Steps 47-52 are typically carried out one sample at a time. Alternatively, multiple samples can be denatured together and then placed on a heat block at 42°C until they are ready to be applied to each microarray.

48. While the sample is denaturing, insert a microarray slide into the bottom portion of a hybridization chamber. Place the hybridization chamber and slide on an inverted heat block at 55°C to prewarm the slide before hybridization.

49. Use compressed air to remove any dust from a LifterSlip. Gently place the LifterSlip onto the prewarmed slide, chamber side toward the glass slide, completely covering the array features area.

50. Quickly centrifuge the denatured sample for a few seconds to collect material in the bottom of the tube. Apply the entire sample to the slide by pipetting slowly along an open edge of the LifterSlip while allowing capillary action to draw the sample under the LifterSlip.
Avoid introducing air bubbles and disrupting the LifterSlip position.

51. Add 15 µL of 3X SSC buffer to each of the two small wells in the bottom portion of the hybridization chamber.
The 3X SSC will keep the chamber humidified during hybridization.

52. Close the hybridization chamber and incubate in a water bath or hybridization oven in the dark at 42°C. Incubate for 12 to 20 h, typically overnight.

Slide Washes

53. Wash and dry the slides using the protocol, solutions, slide racks, and wash chambers found in the NimbleGen Wash Buffer Kit and the NimbleGen Array Processing Accessories Kit. For other array platforms, perform washes as directed by the microarray supplier.
For customized microarrays, wash buffers may need to be optimized, depending on the array design and slide-surface chemistry.

54. Store dried slides in a slide box, protected from light, and preferably in an ozone-free environment. Scan slides as soon as possible.

Slide Scanning and Preliminary Analysis

55. Scan slides according to the description in the NimbleGen Array’s User’s Guide. Save the 532- and 635-nm images as single image .tif files.
See Troubleshooting.

56. Using NimbleScan software, open the scanned image files and create a pair report, as described in the User’s Guide. The pair report generates a GFF file type that can be analyzed further and plotted along chromosomal coordinates using NimbleScan software.

Computational and Statistical Considerations

57. Arrays can be "stripped" and reused with the Nimblegen Array Reuse Kit. However, these arrays show decreases in their M = log(IP/Total) signals with each successive array use (AU). This can cause false positives when attempting to detect variations between experiments whose corresponding AUs differ. To minimize this potential bias, perform comparisons with microarrays subjected to equal numbers of AUs.

58. Following array scanning and Cy3 and Cy5 signal detection, perform array normalization to remove the biases that are typically seen in microarray data.
The characteristics of BrdU-IP-chip experiments render traditional normalization procedures (for RNA-chip and ChIP-chip) suboptimal, so we have designed and implemented normalization software specifically for these data sets (Knott et al. 2009b). Also, we do not recommend traditional ChIP-chip enrichment detection methods for identifying BrdU-enriched genomic regions. These methods typically assume a bimodal M-distribution, which is not the case in BrdU-IP-chip data sets. Instead, we have developed an enrichment detection method specifically for these data sets (Knott et al. 2009b).

TROUBLESHOOTING

Problem: The sample foams excessively during sonication.

[Step 15]

Solution: Consider the following:

1. If foaming occurs, move the tube up the horn so that the microtip reaches the bottom of the (conical) microcentrifuge tube. This usually forces the microbubbles to the top; if not, switch off the sonicator and place the sample on ice.

2. If necessary, briefly centrifuge the sample to collapse the foam.

3. In general, foaming decreases the sonication efficiency and, thus, can reduce shearing of the DNA. Therefore, use the maximum power output that avoids foaming of samples.

Problem: The yield of specifically immunoprecipitated DNA is low.

[Steps 34 and 35]

Solution: As with any procedure involving immunoprecipitation, the quality of the antibody-target interaction is critical to achieving success. Consider the following:

1. Conditions such as lysis buffer composition and wash buffer stringencies may need to be adjusted for specific antibodies.

2. Determine the optimal antibody concentration that maximizes signal-to-noise in a standard BrdU-IP experiment by immunoprecipitating BrdU-labeled DNA with various concentrations of antibody followed by direct PCR analysis of sites known to incorporate and not incorporate BrdU.

3. Alternatively, if chromosomal regions of BrdU incorporation are unknown a priori, examine the total efficiency of BrdU incorporation at different antibody concentrations by immobilizing immunoprecipitated DNA onto nylon membranes (e.g., using a slot-blotter) and probing the membranes with anti-BrdU antibody (Viggiani and Aparicio 2006). This approach can give an indication of the relative amounts of total immunoprecipitated BrdU-labeled DNA, but it does not give an indication of the relative level of nonspecifically immunoprecipitated DNA.

Problem: The amplification reaction does not generate sufficient product.

[Step 38]

Solution: This amplification reaction should yield at least 3 µg of DNA. If necessary, reamplify the samples according to the WGA kit protocol to achieve a higher DNA yield.

Problem: The array fluorescence rapidly degrades.

[Step 55]

Solution: Atmospheric ozone rapidly degrades the fluorescence of the Cy5 dye and can be a major problem. The dyes are especially sensitive after the slides have been dried for scanning. If a persistently weak signal after scanning the slides is noticed, monitor the atmospheric ozone and take precautions to prevent slide exposure.

ACKNOWLEDGMENTS

These studies were supported by NIH grant GM65494 to O.M.A.

REFERENCES

Knott SRV, Viggiani CJ, Tavaré S, Aparicio OM. 2009a. Genome-wide replication profiles indicate an expansive role for Rpd3L in regulating replication initiation timing or efficiency, and reveal genomic loci of Rpd3 function in Saccharomyces cerevisiae. Genes & Dev 23: 1077–1090.[Abstract/Free Full Text]

Knott SRV, Viggiani CJ, Aparicio OM, Tavaré S. 2009b. Strategies for analyzing highly enriched IP-chip datasets. BMC Bioinformatics 10: 305. doi: 10.1186/1471-2105-10-305.[Medline]

Lengronne A, Pasero P, Bensimon A, Schwob E. 2001. Monitoring S phase progression globally and locally using BrdU incorporation in TK(+) yeast strains. Nucleic Acids Res 29: 1433–1442.[Abstract/Free Full Text]

Szyjka SJ, Aparicio JG, Viggiani CJ, Knott S, Xu W, Tavaré S, Aparicio OM. 2008. Rad53 regulates replication fork restart after DNA damage in Saccharomyces cerevisiae. Genes & Dev 22: 1906–1920.[Abstract/Free Full Text]

Vernis L, Piskur J, Diffley JF. 2003. Reconstitution of an efficient thymidine salvage pathway in Saccharomyces cerevisiae. Nucleic Acids Res 31: e120. doi: 10.1093/nar/gng121.[Abstract/Free Full Text]

Viggiani CJ, Aparicio OM. 2006. New vectors for simplified construction of BrdU-incorporating strains of Saccharomyces cerevisiae. Yeast 23: 1045–1051.[Medline]