Analysis of RNA Polymerase II Chromatin Binding by Flow Cytometry

Materials and Reagents

Cell culture and harvest

1. Serological pipettes, polystyrene

2. Sterile conical-bottom tubes, 15 mL

3. Human cervical cancer HeLa Kyoto cells (but other cell types may be used)

4. Penicillin-Streptomycin (P/S) (Thermo Fisher, catalog number: 15140122)

5. Fetal bovine serum (FBS) (Biowest, catalog number: S181B-050)

6. Dulbecco’s modified Eagle medium (DMEM), high glucose, GlutaMAX supplement, pyruvate (Thermo Fisher, catalog number: 31966047)

7. Culture dishes, 6 cm (VWR, catalog number: 353004)

8. EdU (1 μM) (Thermo Fisher, catalog number: A10044)

9. Trypsin 0.25% with EDTA, 100 mL (Life Technologies, catalog number: 25200-056)

10. Phosphate-buffered saline (10× PBS) (Thermo Fisher, catalog number: 70011-051)

11. 10% formalin solution, neutral-buffered ready-made solution (Sigma-Aldrich, catalog number: HT5011)

Extraction

1. HEPES (pH 7.9) (Sigma-Aldrich, catalog number: H3784)

2. MgCl₂ (Sigma-Aldrich, catalog number: 208337)

3. NaCl (Sigma-Aldrich, catalog number: S9888)

4. Sucrose (Sigma-Aldrich, catalog number: S0389)

5. Triton X-100 (Sigma-Aldrich, catalog number: T9284-100ML)

6. PhosSTOP phosphatase inhibitors (Merck, catalog number: 4906837001)

7. cOmplete protease inhibitor cocktail EDTA-free (Merck, catalog number: 5892791001)

8. MG132 (Sigma-Aldrich, catalog number: M7449)

9. ddH₂O Milli-Q water

10. Chromatin extraction buffer (10 mL) (see Recipes)

Staining and preparation of cells for flow cytometry analysis

1. BD Falcon 5 mL polystyrene with cell strainer cap (VWR, catalog number: 734-0001)

2. Aluminum foil

3. Alexa Fluor 647 NHS Ester (Succinimidyl Ester) (Thermo Fisher, catalog number: A20006)

4. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D4540)

5. Phosphate-buffered saline (10× PBS) (Thermo Fisher, catalog number: 70011-051)

6. Fetal bovine serum (FBS) (Biowest, catalog number: S181B-050)

7. Anti-RNAPII (D8L4Y) (Cell Signaling Technology, catalog number: 14958S)

8. Anti-pRNAPII S5 (3E8) (Sigma-Aldrich, catalog number: 04-1572)

9. Anti-pRNAPII S2 (3E10) (Sigma-Aldrich, catalog number: 04-1571)

10. Donkey anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody Alexa Fluor 488 (Thermo Fisher, catalog number: A-21206)

11. Donkey anti-rat IgG H+L Alexa Fluor 488 pre-adsorbed (Abcam, catalog number: ab150153)

12. Click-iT Plus EdU Alexa Fluor 594 Flow Cytometry Assay kit (Thermo Fisher, catalog number: C10646)

13. Bovine serum albumin (BSA) heat-shock fraction, protease free, essentially globulin free, pH 7, ≥98% (Sigma-Aldrich, catalog number: A3059-100G)

14. Na₂HPO₄ (Sigma-Aldrich, catalog number: S3264)

15. KH₂PO₄ (Sigma-Aldrich, catalog number: P5655)

16. KCl (Sigma-Aldrich, catalog number: P3911)

17. NaCl (Sigma-Aldrich, catalog number: S9888)

18. Ethylenediaminetetraacetic acid (EDTA, pH 7.5)

19. Igepal CA-630 (Sigma-Aldrich, catalog number: I8896-50ML)

20. Skim milk powder (Millipore, catalog number: 70166-500G)

21. DNA-stain Hoechst 33258 (624 μg/mL) (Sigma-Aldrich, catalog number: B1155-25MG)

22. LX Alexa Fluor 647 NHS Ester (barcoding) (see Recipes)

23. Flow buffer (1 L) (see Recipes)

24. Detergent buffer (see Recipes)

25. Click-iT permeabilization and wash reagent (see Recipes)

26. Click-iT reaction cocktail (see Recipes)

Equipment

1. Sterile cell culture hood

2. 37 °C cell culture incubator with CO₂ control

3. Centrifuge (Hettich Rotina 380R Centrifuge, rotor 1754, radius 167 mm)

4. LSRII flow cytometer (BD Biosciences), Quartz cuvette flow cell, instrument settings can be found in Table 1.

   
   

   Table 1. LSRII flow cytometer instrument settings

   Parameter	Laser	Band-Pass (nm)	Longpass Dichroic filter (nm)	Detector
   Alexa Fluor 488	488 nm–100 mW	525/30	505	PMT B
   SSC	488 nm–100 mW	488/10	-	PMT C
   FSC	488 nm–100 mW	488/10	-	FSC Diode–Silicon photodiode
   Hoechst	405 nm–100 mW	450/50	-	PMT G
   Alexa Fluor 647	640 nm–40 mW	670/14	-	PMT C
   Alexa Fluor 594	561 nm–45 mW	610/20	600	PMT D

Software

1. DiVa software is used when obtaining flow cytometry data

2. FlowJo V10_5 software is used for analyzing the flow cytometry data

Procedure

Preparations: Prepare the chromatin extraction buffer (see Recipes below); 100 μL is required per sample. As one tablet of cOmplete EDTA-free protease inhibitor cocktail and one tablet of PhosSTOP phosphatase inhibitors are needed per 10 mL solution, adjust with +10 mL if more than 10 mL solution is required. Add MG132 to a final concentration of 20 µM (add 2 µL per milliliter of 10 mM stock). Mix thoroughly by vortexing and place on rotator at 4 °C until use. The chromatin extraction buffer should be prepared fresh before each experiment.

1. Cultivate human cervical cancer HeLa Kyoto cells in DMEM supplemented with 10% FBS and 1% P/S. For 1.0 × 10⁶–1.5 × 10⁶ cells at time of harvest, plate 6.0 × 10⁵ cells in 6 cm culture dishes, approximately 24 h prior to harvest. In addition to dishes intended for samples and the non-treated control cells, include additional dishes intended for use for barcoding and secondary antibody controls. (Approximately 3 × 10⁵ barcoded cells are needed per sample, and 1.0 × 10⁶–1.5 × 10⁶ cells are needed for the secondary controls at time of harvest.)

   Note: The protocol may be adapted for other cell types, e.g., we have used RPE and MRC5 cells.

2. Prepare 1 μM EdU in DMEM supplemented with 10% FBS and 1% P/S at 37 °C and mix well. Remove cell culture media from the culture dishes and replace with 4 mL per dish of the EdU-containing DMEM, 1 h prior to further treatment or harvest in the absence of additional treatment.

   Notes:

   1) Samples intended as barcoding controls and controls for secondary antibodies are not labeled with EdU and are therefore left untreated in this protocol. However, they can be EdU-positive if desired.

   2) This step may be omitted if cell cycle phase determination by DNA stain alone is sufficient.

3. Harvest cells:

   1. Remove culture medium and wash the cell layer once with 500 µL of trypsin solution or PBS at room temperature (RT).

   2. Remove trypsin or PBS solution, add 500 µL of fresh trypsin solution, and transfer the dishes to a 37 °C cell culture incubator. After approximately 1 min, check if cells have started to detach by gently tapping the side of the dish. If the cells still adhere to the surface, place them back in the incubator until detachment is observed.

   3. When most cells have detached, resuspend the cells in 4 mL of DMEM and transfer the cell suspension of each dish to separate and labeled 15 mL sterile conical-bottom tubes.

   4. Isolate cell pellets by centrifugation at 634 × g for 5 min and remove supernatant.

   Note: If timing is important, e.g., after DNA damage, add cold medium to the cells after they have detached, keep the 15 mL sterile conical-bottom tubes containing the cells on ice, and spin the cells at 4 °C to stop enzymatic reactions as much as possible prior to extraction and fixation.

4. Extract the cells: to prepare, place the chromatin extraction buffer on ice (in a Styrofoam box with ice) and the 10% formalin solution at RT.

   1. Add 100 μL of ice-cold chromatin extraction buffer to each cell pellet, mix by pipetting, and immediately place the tube on ice. Start the timer at 5 min after the first sample and then continue with remaining samples. Time is of essence, so work efficiently. If the incubation period exceeds 5 min, then adjust for all samples to obtain equal extraction time.

   2. When time is up, add 900 μL of formalin to the first sample, transfer to RT, and continue with the other samples in the same pace and order as before. Start the timer at 10 min after the last sample has been mixed with formalin and placed at RT.

   3. Isolate cell pellets by centrifugation at 634 × g for 5 min at RT.

   4. Resuspend the cells in 500 µL of PBS and continue with staining.

   Notes:

   1) The extraction is a critical step. Fixation must occur rapidly after extraction, or degradation of RNAPII may occur. Within the time window described here, the time prior to fixation is likely more critical to keep constant than the fixation time itself.

   2) Samples intended as barcoding cells and secondary antibody controls should also be extracted prior to fixation.

   3) When the cells have been fixed and are in PBS, they can be left overnight at 4 °C prior to barcoding and staining.

   4) Prior to barcoding, use PBS without FBS to resuspend the cells, as FBS competes with cellular proteins for binding to Alexa Fluor 647 NHS Ester. However, wash steps with PBS without FBS should be avoided as they lead to loss of cells.

5. Stain cells for barcoding:

   1. Combine cells from tubes containing cells intended for barcoding from step 4d into one tube. (Approximately 3 × 10⁵ barcoded cells should be prepared per sample.)

   2. Add 12.5 μL of LX Alexa Fluor 647 NHS Ester (see Recipes below) per 500 μL of PBS. Mix by inverting the tube and incubate in the absence of light at RT for 30 min.

   3. Quench barcoding by adding 5 mL of PBS with 5% FBS, mix well by inverting the tube, and leave in the dark at RT for 5 min.

   4. Spin down barcoded cells by centrifugation at 1127 × g for 5 min at RT.

   5. Remove supernatant by suction and resuspend pellet containing barcoded cells with 6 mL of PBS with 5% FBS.

   6. Add 5 mL of PBS with 2% FBS to tubes containing samples, secondary antibody controls, and non-treated control cells from step 4d.

   7. Distribute the barcoded cells from step 5e equally between all tubes containing samples, secondary antibody controls, and non-treated control cells from step 5f (e.g., for 10 tubes, distribute 6 mL of barcoded cell solution/10 = 0.6 mL of barcoded cell suspension per tube).

6. Stain samples with primary and secondary antibodies

   1. Spin down cells in tubes, now also containing barcoding cells, by centrifugation at 1127 × g for 5 min at RT. Remove most of the supernatant by suction (until approximately 100 μL is left), spin down again, and remove rest of the supernatant with a 200 μL pipette.

      Note: The second spin is to ensure the staining is as equal as possible between the samples. If this step is omitted, remaining supernatant adhered to the sides of the 15 mL tube may fall into the staining solution and affect the final staining volume, and thus antibody concentrations during staining.

   2. Resuspend pellet in 50 μL of detergent buffer (see Recipes) and leave at RT for 5 min.

   3. Add 50 μL of detergent buffer with primary antibody: rabbit RNAPII NTD 1:50 (i.e., final dilution 1:100), rat pRNAPII S5 1:50 (i.e., final dilution 1:100), or rat pRNAPII S2 1:50 (i.e., final dilution 1:100). Incubate in the absence of light at RT for 1 h.

      Notes:

      1) The tube with cells intended for use as secondary antibody control is resuspended in 100 μL of detergent buffer without primary antibody.

      2) If EdU staining is included, each sample can be stained with one antibody only.

   4. Add 5 mL of PBS with 2% FBS to each tube.

   5. Spin down cells by centrifugation at 1127 × g for 5 min at RT. Remove most of the supernatant by suction (until approximately 100 μL is left), spin down again, and remove rest of the supernatant with a 200 μL pipette.

   6. Add 100 μL of detergent buffer with secondary antibody: anti-rabbit Alexa Fluor 488 1:500 or anti-rat Alexa Fluor 488 1:500. Incubate in the absence of light at RT for 30 min.

7. Label EdU with the Click-iT Plus EdU Alexa Fluor 594 Flow Cytometry Assay kit. Prepare by thawing the reaction buffer additive (10×), fluorescent dye azide 594, and copper protectant.

   1. Add 5 mL of PBS with 2% FBS to each tube.

   2. Spin down cells by centrifugation at 1127 × g for 5 min at RT. Remove most of the supernatant by suction (until approximately 100 μL is left), spin down again, and remove rest of the supernatant with a 200 μL pipette.

   3. Resuspend cell pellet in 100 μL of 1× Click-iT permeabilization and wash reagent (see Recipes) and leave for 15 min at RT.

   4. Prepare Click-iT reaction cocktail (see Recipes). This should be used within 15 min of preparation.

   5. Add 500 μL of Click-iT reaction cocktail and incubate in the absence of light at RT for 30 min.

   6. Add 5 mL of PBS with 2% FBS to each tube.

   7. Spin down cells by centrifugation at 1127 × g for 5 min at RT. Remove most of the supernatant by suction (until approximately 100 μL is left), spin down again, and remove rest of the supernatant with a 200 μL pipette.

8. Label DNA with Hoechst 33258.

   1. Resuspend cells in 500 μL of flow buffer (see Recipes) containing 2.4 μL/mL Hoechst 33258. Incubate in the absence of light at RT for 15 min.

   2. Store in the absence of light at 4 °C overnight or until running flow cytometry.

Data analysis

1. Acquire data on the flow cytometer

   1. In the flow cytometer acquisition software (DiVA), select the parameters shown in Table 2.

      
      

      Table 2. Flow cytometry acquisition parameters

      Parameter	Scale
      Side scatter (SSC)	linear
      Forward scatter (FSC)	linear
      Hoechst-W (width)	linear
      Hoechst-A (area)	linear
      Alexa Fluor 647	log
      Alexa Fluor 594	log
      Alexa Fluor 488	log

      
      

   2. Open the graphs shown in Table 3.

      
      

      Table 3. Flow cytometry acquisition graphs

      Output	x-axis	y-axis
      Size and granularity of cells	FSC	SSC
      Single cells	Hoechst-W	Hoechst-A
      Barcoding	Alexa Fluor 594	Alexa Fluor 647
      DNA profile	Hoechst-A	histogram
      DNA vs. RNAPII/pRNAPII S5/pRNAPII S2	Hoechst-A	Alexa Fluor 488
      EdU profile	Hoechst-A	Alexa Fluor 594

   
   

   3. Adjust voltages for FSC vs. SSC such that the majority of the cell population is within the graph window.

   4. Adjust voltage for Hoechst such that the first peak of the histogram is located at 50 K (corresponds to G1 phase).

   5. Gate for single cells based on the distribution of cells in the Hoechst-W vs. Hoechst-A plot (see Figure 2A). While the Hoechst-A will increase during the cell cycle as the DNA content doubles in size, the Hoechst-W (corresponding to the time it takes for the cell to traverse the laser beam) will not increase as much. Doublets (e.g., two G1 cells clumped together) may have equal Hoechst-A signal as G2 cells and can contaminate the G2 cell population. Bu using a narrow Hoechst-W gate, doublets are removed from analysis, as they will have a larger Hoechst-W than single cells (because they take longer time to traverse the laser beam).

   6. Separate barcoded cells (ctrl, shown in orange) from sample (shown in black) based on Alexa Fluor 647 signal and EdU incorporation (Alexa Fluor 594) (Figure 2B). Note that there is bleed-through from high EdU signal into the Alexa Fluor 647 channel, as a subset of the sample population with high Alexa Fluor 594 staining also show high Alexa Fluor 647 intensity. Nevertheless, the barcoded cells form a distinct population that can be separated from the sample cells. Importantly, there is no bleed-through from the EdU (Alexa Fluor 594 signal) into the RNAPII signal (Alexa Fluor 488) (Figure 3).

   7. Gate for cell cycle phases based on EdU incorporation vs. DNA content (Figure 2Ci): G1 cells are negative for EdU and have G1 DNA content. Early S-phase cells have DNA content close to G1 but are EdU positive. Mid S have an intermediary DNA content and are EdU positive. Late S phase have similar DNA content as G2 cells but are EdU positive. G2 cells are EdU negative and have G2 DNA content. Mitotic cells are observed as the small population below the G2 cells and are not included in the analysis of G2 cells. Alternatively, gate for cell cycle phases by DNA content (Figure 2Cii): a strict G1 gate based on DNA content is performed by selecting only the cells to the left of the summit of the G1 peak. This strict gate will minimize contamination of S-phase cells. Likewise, G2 cells are selected to the right of the summit of the G2 peak to minimize contamination of S-phase cells. The S-phase gate should be located between the G1 and G2 peaks. Choosing a location well separated from these will minimize contamination of G1 and G2 cells. The cell cycle gates should be as equal as possible in all the samples during analysis.

   8. Determine median RNAPII levels in individual cell cycle phases.

   9. Raw data output files are generated as .fcs files.

      Acquire 20,000 cells of the population identified as sample after separating the sample from the barcoded cells (Figure 2B). If the number of cells is limiting, the minimum number of cells acquired should be 5,000. Use either slow or medium speed when acquiring data on the flow cytometer. Keep the speed constant for all samples.

      
      

      
      Figure 2. Gating strategy of flow cytometry data. A. Adjust Hoechst voltage so that the G1 peak summit is located at 50 K and gate for single cells as shown. B. Separate the barcoded cells from the sample cells by Alexa Fluor 647 vs. Alexa Fluor 594 signal as shown. C. Gate for cell cycle phases in the sample as indicated based on EdU incorporation and DNA content (Hoechst-A) as shown (i) or DNA content alone (ii) (see text for more details). D. Analyze RNAPII levels in the entire sample, and in individual cell cycle phases, by obtaining the median levels from the cell cycle phase populations defined in C. The cells in the different cell cycle phases from C are shown in the RNAPII scatter plots in D.

      
      

      
      Figure 3. No bleed-through from Alexa Fluor 594-A (EdU) into Alexa Fluor 488-A (RNAPII) measurements. Scatter plots showing A) Alexa Fluor 594 (EdU) and B) Alexa Fluor 488 (RNAPII) intensities vs. Hoechst-A in sample and barcoded cells. Sample cells were identical to the barcoded cells, with the exception of EdU incorporation (performed on sample cells only) and Alexa Fluor 647 barcoding (performed on barcoded cells only). Sample cells show high Alexa Fluor 594 intensity in S phase compared to barcoded cells, which were not treated with EdU. However, intensities of the sample cells overlap completely with the intensities of the barcoded cells in the Alexa Fluor 488 scatter plot, showing that there is no bleed-through from Alexa Fluor 594 into the Alexa Fluor 488 measurements using the conditions described in this protocol.




2. Analysis of the flow cytometry data

   1. Import .fsc files into a workspace on the flow cytometer software FlowJo V10_5 or other versions of the software.

   2. Use the gating strategy depicted in Figure 2.

   3. RNAPII profiles can be viewed in Layout Editor and median RNAPII levels can be determined in Table Editor.

   4. RNAPII level in the sample is determined by subtracting the background fluorescence (using the median level of Alexa Fluor 488 staining in the whole secondary antibody control sample) and by normalizing to the median level of Alexa Fluor 488 staining in the whole barcoded cell sample (Equation 1, G1 levels are shown as an example of cell cycle phase). Also include non-treated cells as one of the samples. This should be used to measure changes after treatment (Equation 2, G1 levels are shown as an example).

      
      

      

Recipes

1. Chromatin extraction buffer (10 mL)

   Reagent	Stock	Final concentration	Volume
   HEPES, pH 7.9	1 M	20 mM	200 μL
   MgCl2	0.1 M	1.5 mM	150 μL
   NaCl	5 M	140 mM	280 μL
   Sucrose	2.5 M	300 mM	1,200 μL
   Triton X-100	100%	0.5%	50 μL
   ddH2O Milli-Q water			Adjust with water up to total 10 mL

   Prior to use, add one tablet of cOmplete EDTA-free protease inhibitor cocktail and one tablet of PhosSTOP phosphatase inhibitors per 10 mL of solution. Add 2 μL of 10 mM MG132 per milliliter of buffer for a final concentration of 20 μM. The extraction buffer should be prepared fresh the day of extraction and kept at 4 °C or on ice prior to use.




2. LX Alexa Fluor 647 NHS Ester (barcoding)

   Add 2 μL of Alexa Fluor 647 NHS Ester (5 mg/mL) stock in 553.5 μL of DMSO; the resulting solution is named working solution (18 μg/mL). Dilute the working solution further by combining 200 µL of the working solution with 160 µL of DMSO. The final solution is called the LX solution (10 µg/mL). Once diluted, the working solution and the LX solution can be stored at -80 °C and are stable for several weeks.




3. Flow buffer (1 L)

   Reagent	Stock	Final concentration	Volume
   Na2HPO4	0.25 M	6.5 mM	26 mL
   KH2PO4	1 M	1.5 mM	1.5 mL
   KCl	1 M	2.7 mM	2.7 mL
   NaCl	5 M	137 mM	27.4 mL
   EDTA, pH 7.5	0.5 M	0.5 mM	1 mL
   ddH2O Milli-Q water			Adjust with water up to total 1 L

   Add 100 μL of Igepal CA-630 100% to 100 mL of flow buffer to obtain a final concentration of 0.1%.




4. Detergent buffer

   Flow buffer + 4% skim milk powder. For 4 mL of flow buffer, add 160 mg of skim milk powder.




5. Click-iT permeabilization and wash reagent

   Reagent	Volume
   PBS	4.5 mL
   Component E	500 μL
   BSA	1%

   Component E is from the Click-iT Plus EdU Alexa Fluor 594 Flow Cytometry Assay kit.




6. Click-iT reaction cocktail

   The copper protectant, fluorescent dye picolyl azide, and 10× reaction buffer additive are obtained from the Click-iT Plus EdU Alexa Fluor 594 Flow Cytometry Assay kit. Dilute 10× reaction buffer additive 1:10 in ddH₂O Milli-Q water.

   Reagent	Volume (for one reaction/sample)
   PBS	438 μL
   Copper protectant (Component F)	10 μL
   Fluorescent dye picolyl azide	2.5 μL
   Reaction buffer additive 1×	50 μL
   Total reaction volume	500 μL

   Adjust volumes according to number of samples in the experiment.

Acknowledgments

The method described here is further developed from Landsverk et al. (2020), Håland et al. (2015) and Stokke et al. (1993). We would like to thank the flow cytometry core facility at the Radium Hospital, Oslo University Hospital, for assistance and use of the flow cytometers. This work was funded by the Norwegian Research Council (275918) and Nansenfondet og de dermed forbundne fond.

Competing interests

The authors declare no competing interests.

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