TITLE: Proximity Ligation Assay to Detect Transcription-Replication Conflicts in Yeast
AUTHORS: Rebecca E Brown, Catherine H Freudenreich

[DESCRIPTION]
Replication and transcription machineries can come into conflict with each other when spatial and temporal separation of these processes are not possible. We present a protocol to examine transcription-replication conflicts in the budding yeast Saccharomyces cerevisiae. We have modified existing proximity ligation assay protocols to detect proximity between RNA polymerase II (transcription machinery) and PCNA (replisome) in yeast. This protocol can be applied to any two proteins or modifications of interest in which you have antibodies suitable for immunofluorescence.

   
This proximity ligation assay (PLA) protocol was modified from Alberts et al. JOVE 2019 with the following changes:
Different antibodies targeting RNA polymerase II and PCNA instead of cytosolic chaperone proteins
Using nocodazole as a negative control (inhibiting replication by arresting cells in M phase)
Altered wash conditions and slides used
Added details about imaging and image analysis (using Cell Profiler)
 

Cell Profiler can be used for quantifying foci per nucleus. Pipelines used are included for download and details about image analysis are shown below in the steps of the protocol.
 




[BEFORE_START]
1. Make all solutions before starting the protocol:

Nocodazole:
Make 1.5 mg/mL nocodazole solution in DMSO. Dilute to 15 µg/mL concentration in yeast rich liquid media. 

4% Paraformaldehyde (PFA):
Dissolve 0.8 g PFA in pre-warmed 17 mL 0.1 M Na2HPO4.
Add 3 mL of 0.1 M NaH2PO4. 
Final pH at 7.6.

Wash buffer:
1.2 M Sorbitol in 100 mM KPO4 pH 6.5

Zymolyase solution:
0.5 mg/mL Zymolyase 100 T, 1.2 M Sorbitol, 100 mM KPO4 pH 6.5, 20 mM 2-Mercaptoethanol.
Make fresh for each experiment. 

Permeabilizing solution:
1% TritonX-100 in 100 mM KPO4 pH 6.5.

Wash buffer A:
Make according to Duolink® manufacturer guidelines.

Wash buffer B:
Make according to Duolink® manufacturer guidelines.


2. Prepare humidity chamber (see step 18). 

[STEPS]
SECTION: Preparation of Poly-L-Lysine Coated Slides
1. Prepare poly-L-lysine coated polytetrafluoroethylene (PTFE) slide to ensure that yeast cells adhere to slide:

NOTE: Slides should be prepared either day of or day before proximity ligation assay protocol is started.

NOTE: Be sure not to touch the wells of the slide for all remaining steps of this protocol. Make sure to label the slides with pencil only.
SECTION: Cell Growth and Collection
6. NOTE: For all centrifugation steps, make sure to spin at low speeds (<3000 rpm is recommended).
SECTION: Cell Fixation
10. Fix cells with 4 % (m/v) paraformaldehyde (PFA) for 15 min at room temperature. Use 100 µL of 4 % (m/v) PFA per 1 mL of cells collected. Collect cells by centrifugation at 3000 rpm for 5 min after incubation in PFA.
SECTION: Cell Wall Digestion to Prepare Spheroplasts
12. Cell wall is degraded through enzymatic digestion by Zymolyase:

Digest cell walls with Zymolyase solution (0.5 mg/mL  Zymolyase 100 T, 1.2 molar (M)  sorbitol, 100 millimolar (mM) KPO4 pH 6.5, 20 millimolar (mM) 2-Mercaptoethanol) at 30 °C for 20 min.

NOTE: Treat spheroplasts carefully to prevent disruption of nuclear structure. 
SECTION: Attaching Spheroplasts to Slide
15. Resuspend spheroplasts in 30 µL - 100 µL of 1.2M sorbitol. Attach spheroplasts to poly-L-lysine coated slides by pipetting 5 µL into each well. Let spheroplasts sit for 30 min at Room temperature before aspirating liquid and proceeding with next steps.

NOTE: Altering volume of sorbitol to suspend spheroplasts may need to be altered to optimize the density of cells upon the slide while imaging.
SECTION: Cell Permeabilization
16. Wash cells 3x in with wash buffer (1.2 molar (M) sorbitol in 100 millimolar (mM) KPO4 pH 6.5). Use 5 µL of wash buffer per well. Let wash buffer sit on well for 5 min. Aspirate liquid using a pipette tip connected to a vacuum source after each wash. 

NOTE: Make sure not to touch wells when aspirating solution. Touch tip to the edge of the well to aspirate the solution. 
SECTION: Proximity Ligation Assay (PLA) - Blocking
18. The steps in the PLA sections below follow the Duolink® PLA Fluorescence Protocol (Duolink PLA Fluorescence Protocol) using reagents from the kit provided by Millipore Sigma (see materials).

NOTE: Before proceeding with steps, prepare a humidity chamber and equilibrate to 37 °C. 

 
SECTION: PLA - Primary Antibody Incubation
22. Vortex Duolink® Antibody Diluent.
SECTION: PLA - PLA Probe Incubation
27. NOTE: Heat up humidity chamber to 37 °C before proceeding with these steps.

While chamber is heating up: Vortex PLUS and MINUS PLA probes and Duolink® Antibody Diluent.
SECTION: PLA - Ligation
34. NOTE: Wait to add Ligase to solution until immediately prior to addition to sample. Make sure ligation buffer is completely thawed/mixed prior to use. Ligation buffer can be aliquoted to prevent multiple freeze/thaw cycles. 

Make 1X ligation buffer solution: Dilute the 5X Duolink® Ligation buffer 1:5 in molecular biology grade water. 
For each 5 µL reaction: add 1 µL of 5X Duolink® Ligation buffer and 4 µL of molecular biology grade water.
SECTION: PLA - Amplification
41. NOTE: Wait to add Polymerase until immediately prior to addition to sample. Amplification buffer is light-sensitive (protect all solutions containing buffer from light). 

Make 1X amplification buffer solution: Dilute the 5X Duolink® Amplification buffer 1:5 in molecular biology grade water. 
For each 5 µL reaction: add 1 µL of 5X Duolink® Amplification buffer and 4 µL of molecular biology grade water.
SECTION: PLA - Final Slide Preparation
48. NOTE: Light sensitive reagents - keep slides protected from light at all times. 

Aspirate Amplification solution from slide wells.

NOTE: Make sure not to touch wells when aspirating solution. Touch tip to the edge of the well to aspirate the solution. 

SECTION: Image Analysis
55. Quantification of foci per nucleus can be done by manual counting, using Cell Profiler, or other available software. Below is analysis done with Cell Profiler: 
SECTION: Preparation of Poly-L-Lysine Coated Slides
2. Gently scrub PTFE slide using toothbrush with soft bristles (i.e. baby toothbrush).
SECTION: Preparation of Poly-L-Lysine Coated Slides
3. Rinse slide with deionized water and let slide dry completely in hood for 15 min.
SECTION: Preparation of Poly-L-Lysine Coated Slides
4. Place 5 µL of 0.1% poly-L-lysine onto each well and let sit for 10 min at Room temperature.
SECTION: Preparation of Poly-L-Lysine Coated Slides
5. Rinse slide with deionized water and let slide dry completely in hood (for at least 60 min at Room temperature, overnight is acceptable).
SECTION: Cell Growth and Collection
7. Grow cells overnight at 30 °C to saturation (use roller drum or shaking incubator) and dilute back to an OD600 ~0.2.

NOTE: Cells can be grown in media of choice. For this protocol, yeast synthetic complete dropout media was used for all experiments except for those treating cells with nocodazole, in which cells were grown in yeast rich media instead.
SECTION: Cell Growth and Collection
8. Grow cells to an OD600 ~1.5.

NOTE: Growth time will vary per strain (typically ~4-10 hrs after diluting back to OD600 ~0.2). 
SECTION: Cell Growth and Collection
9. Collect cells by centrifugation at 3000 rpm for 5 min. Collect 1 mL of cells per condition (i.e. for each suspension of prepared spheroplasts in sorbitol - see step 15).

NOTE: Volume and OD600 of cells collected may need to be altered to optimize the density of cells upon the slide while imaging.
SECTION: Cell Growth and Collection
8.1. For experiments treating cells with nocodazole, grow cells to OD600 ~1.0. Add nocodazole (15 µg/mL final concentration in cultures) and grow cells for 60 min before collection.

NOTE: An important negative control to include is a sample that does not express one of the targets (in this case PCNA and RNA polymerase II) to ensure that primary antibodies are specific. Since PCNA and RNAPII are essential proteins, replication or transcription must be inhibited as a negative control and should result in reduced PLA foci. Here we chose to inhibit replication by treating cells with nocodazole to arrest cells in M phase. 
SECTION: Cell Fixation
11. Wash cells 3x in with wash buffer (1.2 molar (M) sorbitol in 100 millimolar (mM) KPO4 pH 6.5). Use 100 µL of wash buffer per 1 mL of cells collected. Pellet cells between washes by centrifugation at 3000 rpm for 5 min.
SECTION: Cell Wall Digestion to Prepare Spheroplasts
13. Spin down cells at 2000 rpm for 5 min  to remove Zymolyase solution.
SECTION: Cell Wall Digestion to Prepare Spheroplasts
14. Wash cells 3x in with wash buffer (1.2 molar (M) sorbitol in 100 millimolar (mM) KPO4 pH 6.5). Use 100 µL of wash buffer per 1 mL of cells collected. Pellet cells between washes by centrifugation at 2000 rpm for 5 min.
SECTION: Cell Permeabilization
17. Wash attached spheroplasts 3x with permeabilizing solution (1 % (v/v) TritonX-100 in 100 millimolar (mM) KPO4 pH 6.5). Use 5 µL of permeabilizing solution per well. Let permeabilizing solution sit on well for 5 min. Aspirate liquid using a vacuum source after each wash.
SECTION: Proximity Ligation Assay (PLA) - Blocking
19. Vortex the Duolink® Blocking Solution.
SECTION: Proximity Ligation Assay (PLA) - Blocking
20. Add 5 µL of Blocking Solution to each sample well (be sure to cover entire sample with Blocking Solution).
SECTION: Proximity Ligation Assay (PLA) - Blocking
21. Incubate slide in heated humidity chamber for 60 min at 37 °C.
SECTION: Image Analysis
56. Three dimensional images need to be split into separate channels, positions, and timepoints using the following code:

bfconvert imagename.file_ending imagename_C%c_P%s_T%t.tif
SECTION: Image Analysis
57. Use Cell Profiler to create maximum intensity projections of images. Pipeline used is linked here and can be downloaded/modified for future use. 
  

See directions and example screenshots below:

SECTION: Image Analysis
57.5. Use MakeProjection module to create maximum intensity projection images:
  
SECTION: Image Analysis
58. Use Cell Profiler to quantify foci per nucleus. Pipeline used is linked here and can be downloaded/modified for future use. 
  

See directions and example screenshots below:
SECTION: Image Analysis
57.6. Use SaveImages module to save maximum intensity projection images:
 
SECTION: Image Analysis
58.3. Use IdentifyPrimaryObjects module to identify nuclei:
  
  
SECTION: Image Analysis
58.4. Use IdentifyPrimaryObjects module to identify foci:
  
  
SECTION: Image Analysis
58.5. Use RelateObjects module to group together nuclei and PLA foci (this will allow for identification of only foci within nuclei):
  
SECTION: Image Analysis
58.6. Use ExportToSpreadsheet module to export quantification of foci per nucleus:
  
  

SECTION: PLA - Primary Antibody Incubation
23. Dilute your primary antibodies to suitable concentration in Duolink® Antibody Diluent. The following antibodies/dilutions were used:
1:400 dilution of anti-PCNA [5E6/2] (abcam ab70472)
1:400 dilution of anti-RNAPII [p Ser2] (Novus Biologicals NB100-1805)

NOTE: Antibody dilutions may need to be optimized if using different antibodies or for different strains/growth conditions. 
SECTION: PLA - Primary Antibody Incubation
24. Aspirate Duolink® Blocking Solution from slide wells. 

NOTE: Make sure not to touch wells when aspirating solution. Touch tip to the edge of the well to aspirate the solution. 
SECTION: PLA - Primary Antibody Incubation
25. Add diluted primary antibody solution to each well as stated below:
Experimental condition: create mix of the two primary antibody dilutions (2.5 µL of each antibody dilution per well). Add 5 µL of mixed primary antibodies to each well, making sure to cover the entire sample. 
Single antibody controls: Add 5 µL of individual primary antibody to each well, making sure to cover the entire sample. 

NOTE: An important control to include with each PLA experiment is single antibody controls. Adding only one antibody to the reaction should eliminate or at least drastically decrease the amount of PLA foci detected. 
SECTION: PLA - Primary Antibody Incubation
26. Incubate slides in a humidity chamber at 4 °C overnight. 

NOTE: Optimal incubation temperature/time for primary antibodies chosen will need to be determined.

SECTION: PLA - PLA Probe Incubation
28. Make PLA probe solution: Dilute the PLUS/MINUS PLA probes 1:5 in Duolink® Antibody Diluent. 
For each 5 µL reaction: add 1 µL of each probe and 3 µL of Duolink® Antibody Diluent. 
SECTION: PLA - PLA Probe Incubation
29. Aspirate primary antibody solution from slide wells. 

NOTE: Make sure not to touch wells when aspirating solution. Touch tip to the edge of the well to aspirate the solution. 
SECTION: PLA - PLA Probe Incubation
30. Wash slide wells 2x for 5 min in 1x Wash Buffer A at Room temperature. Let wash buffer sit on slide and aspirate solution between each wash. 
SECTION: PLA - PLA Probe Incubation
32. Apply 5 µL of PLA probe solution to each slide well, making sure to cover the entire sample. 
SECTION: PLA - PLA Probe Incubation
33. Incubate slides in pre-heated humidity chamber for 60 min at 37 °C.
SECTION: PLA - Ligation
35. Aspirate PLA probe solution from each slide well.

NOTE: Make sure not to touch wells when aspirating solution. Touch tip to the edge of the well to aspirate the solution. 
SECTION: PLA - Ligation
36. Wash slide wells 2x for 5 min in 1x Wash Buffer A at Room temperature. Let wash buffer sit on slide and aspirate solution between each wash. 
SECTION: PLA - Ligation
37. Make Ligase solution: During the wash steps retrieve Ligase from freezer using freezer block. Add Ligase to 1x ligation buffer at a 1:40 dilution.
For each 5 µL reaction: add 0.125 µL of Ligase and 4.875 µL of 1x ligation buffer.
SECTION: PLA - Ligation
38. Aspirate excess Wash Buffer A from slide wells.
SECTION: PLA - Ligation
39. Apply 5 µL of Ligase solution to each slide well, making sure to cover the entire sample.  
SECTION: PLA - Ligation
40. Incubate slides in pre-heated humidity chamber for 30 min at 37 °C.
SECTION: PLA - Amplification
42. Aspirate Ligase solution from each slide well.

NOTE: Make sure not to touch wells when aspirating solution. Touch tip to the edge of the well to aspirate the solution. 
SECTION: PLA - Amplification
43. Wash slide wells 2x for 2 min in 1x Wash Buffer A at Room temperature. Let wash buffer sit on slide and aspirate solution between each wash. 
SECTION: PLA - Amplification
44. Make Amplification solution: During the wash steps retrieve Polymerase from freezer using freezer block. Add Polymerase to 1x amplification buffer at a 1:80 dilution.
For each 5 µL reaction: add 0.0625 µL of Ligase and 4.9375 µL of 1x amplification buffer.
SECTION: PLA - Amplification
45. Aspirate excess Wash Buffer A from slide wells.
SECTION: PLA - Amplification
46. Apply 5 µL of Amplification solution to each slide well, making sure to cover the entire sample.  
SECTION: PLA - Amplification
47. Incubate slides in pre-heated humidity chamber for 100 min at 37 °C.
SECTION: PLA - Final Slide Preparation
49. Wash slide wells 2x for 10 min in 1x Wash Buffer B at Room temperature. Let wash buffer sit on slide and aspirate solution between each wash. 
SECTION: PLA - Final Slide Preparation
50. Wash slides once in 0.01x Wash Buffer B for 1 min.
SECTION: PLA - Final Slide Preparation
51. Aspirate excess Wash Buffer B from slide wells. 
SECTION: PLA - Final Slide Preparation
52. Mount the slides with a coverslip using a minimal volume of Duolink® PLA Mounting Medium with DAPI. Make sure to leave space on slide around coverslip to allow for sealing with nail polish. Seal edges of coverslip to slide with clear nail polish. 
SECTION: PLA - Imaging
54. Image samples using an oiled 100x objective. See below for details imaging on a Leica Dmi8 Thunder or DeltaVision Ultra widefield fluorescence microscope with Duolink® In Situ Red Detection Reagents:
SECTION: PLA - Imaging
54.1. Using Leica Dmi8 Thunder:
For imaging:
Use 575nm excitation for PLA foci
Use 395nm excitation for DAPI

For deconvolution:
Small Volume Computational Clearing (Under Thunder/Lightning in Process tab)
Adaptive strategy
Refractive Index/Mounting Medium - 1.461/custom
SECTION: PLA - Imaging
54.2. Using DeltaVision Ultra:
For imaging:
Use Red channel for PLA foci
Use Blue channel for DAPI
Used 1.5180 Laser Liquid Oil for use with objective requiring oil

For deconvolution:
Add deconvolution to processing

SECTION: PLA - Final Slide Preparation
53. Wait at least 15 min and up to 2 days before imaging samples using a fluorescence or confocal microscope. For storage of 1-2 days, keep slide in the dark at 4 °C. Slide can be stored longer term at -20 °C.

SECTION: Image Analysis
57.1. Use Images module to compile list of files to be analyzed:
  
SECTION: Image Analysis
57.2. Use MetaData module extract data from file names:
 
   
SECTION: Image Analysis
57.3. Use NamesAndTypes module to assign names to image files:
  
SECTION: Image Analysis
57.4. Use Groups module to group files by name of file and channel used:
  
SECTION: Image Analysis
58.1. Use MetaData module extract data from file names:
  
SECTION: Example of Data
59. Data shown is from Brown et al. PLOS Biology 2022. 

   
  
  
SECTION: Image Analysis
58.2. Use NamesAndTypes module to assign names to image files:
  
  
SECTION: PLA - PLA Probe Incubation
31. Aspirate excess Wash Buffer A from slide wells.