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1. Imaging of EWS-FLI1 condensation formation on DNA Curtains
- Open the imaging software, find and mark the positions of the 3 × 3 zig-zag patterns under the bright field.
- Turn on the flow at 0.2 mL/min to stain the DNA with the double-stranded DNA dye for 10 min.
- Dilute mCherry-EWS-FLI1 with the imaging buffer to the concentration of 100 nM in 100 µL in the tube.
- Load the protein sample through the valve with a 100 µL glass syringe, and change the flow rate to 0.4 mL/min.
- Turn on the 488 nm laser and pre-scan each region to check the DNA distribution state; select the region where the DNA molecules distribute evenly. Set the laser power to 10% for the 488 nm laser and 20% for the 561 nm laser. Use the power meter to measure the real laser power near the prism: 4.5 mW for the 488 nm laser and 16.0 mW for the 561 nm laser.
- Image acquisition
- Start acquiring images at 2 s intervals with both 488 nm and 561 nm lasers simultaneously.
- Change the valve from manual to injection mode to let the imaging buffer flush the protein sample into the flow cell after 60 s.
NOTE: This process will take ~30 s, and the field of view will be covered with mCherry signals as soon as the EWS-FLI1 proteins reach the flow cell.
- To remove free EWS-FLI1, keep washing the flowcell with the imaging buffer for 5 min with only the 561 nm laser switched on. Stop the flow and incubate at 37 °C for 10 min.
- Turn on the flow at 0.4 mL/min to let the DNA extend, and acquire images at 2 s intervals between different frames to obtain high-throughput data of EWS-FLI1 condensate formation.
2. Intensity analysis for mCherry-EWS-FLI1
- Import the data as image sequences into ImageJ software, pick out the puncta at 25× GGAA sites in an 8×8 pixels square, and save the image in .tif format.
- Calculate the intensity as the summation of the intensity in the 8×8 pixels square, including the whole puncta, and remove the background by subtracting the intensity of the background in an area of the same size near the 8x8 pixels square.