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DNA Combing Protocol

Protocols

Kavi's protocol, January, 2022:

Combing-Protocol-01-2022

Kavi's protocol January, 2019:

Combing-Protocol-01-2019

Other protocols:

Genomic Vision Protocol: RCA_Protocol_September_2017_M

STARprotocols published protocol 2022: https://www.sciencedirect.com/science/article/pii/S2666166722002519

Word version of Sarah's protocol:

Combing Protocol

Pre-Day 1: Split cells into a 6 well plate (or 35 mm dish) so that they will be about 50-70% confluent the next day. Cells cannot become too confluent or they will not be cycling. Equilibrate a flask of media overnight to use for dilutions the next day. A flask of HBSS should be equilibrated overnight as well.

Day 1 – Need heat blocks or water baths at 68C and 50C, will need 50C overnight.

  1. Treat cells with 20 µM IdU for 20 minutes.
    1. All dilutions should be done using equilibrated media.
    2. IdU is resuspended in 1 N ammonium hydroxide at 50 mg/mL or 141.2 mM
    3. IdU and CldU are in 20 µL aliquots, stored at -20
  2. Rinse 2x equilibrated HBSS (2 mLs per rinse).
  3. Treat with 100 µM CldU for 20 minutes.
    1. CldU is resuspended in 1 N ammonium hydroxide at 20 mg/mL or 76.1 mM
  4. During CldU treatment (or earlier), place Buffer 2 (agarose for plugs) at 68C for at least 10 min to melt it, then place at 50C to keep warm.
  5. Wash the wells 2x with equilibrated HBSS.
  6. Trypsinize the cells at Room Temperature (300 µL/well for a 6-well plate).
  7. Use ~500 µL media to suspend cells and add to eppendorf tube. Spin cells down at 2000 rcf for 2 minutes.
  8. Aspirate media and resuspend cells in ice cold PBS.
    1. Usually ~500 uL of PBS for RPE cells.
  9. Determine the cell concentration for each condition.
  10. Transfer 150,000 cells (or appropriate number for the cell line you’re using) to a new Eppendorf tube.
    1. If you have less than the desired cell number, you can still proceed but will just have a lower density of fibers on the coverslip.
    2. 150,000 cells is good for RPE. ~100,000 cells for U2OS.
  11. Pellet cells at 150 rcf for 5 minutes (very slow, not a typo)
  12. Pipette off PBS and add 45uL of Buffer 1. Homogenize well by pipetting – do not want any clumps of cells.
  13. Place at 50C for at least 10 seconds to warm cell solution.
  14. Add 45uL of Buffer 2 and pipette up and down at 50C to homogenize.
  15. Pipette all 90uL into DNA plug molds
  16. Place the plug mold in a freezer box (careful that it does not touch the top when closed) and put at 4C for 30min
    1. Can leave the plugs at 4C for several hours if needed
  17. For each plug, mix 225uL Buffer 3 with 25uL Component 3 in an Eppendorf tube
    1. Must be made fresh
  18. Remove tape from the bottom of the plug mold and use the plunger from the end of the mold to push the solidified plugs into the Buffer 3/Component 3 solution. Make sure plugs are completely immersed.
    1. Easiest to push plugs out from the bottom
  19. Place at 50C for 30 min
  20. Invert the tubes gently a few times to mix, then leave at 50C overnight.

Day 2 – Need heat blocks or water baths at 68C and 42C toward the end of the day, will need 42C overnight. If using water baths, will need to use aluminum block to stabilize tubes.

  1. Dilute Buffer 4 1:100 with nuclease free water. Will need 45mL per plug.
    1. Need to make fresh
  2. Pour off majority of liquid from plugs (plug should stick to side of tube) and transfer to 15mL conical filled with diluted Buffer 4.
    1. Important to use 15mL of buffer in each tube to avoid breaking the plugs during rotation.
    2. Transfer plug to tube after 15mL of buffer has been added, not before.
  3. Rotate at room temp for at least 1 hour
  4. Pipette off majority of buffer (plugs should settle to the bottom of the tube), being careful not to pipette too close to the plug as they are very delicate. Can leave 1-2mL of buffer in the bottom of the tube.
  5. Slowly add another 15mL of diluted Buffer 4 down the side of the tube. Rotate 1+ hour.
  6. Repeat steps 4 & 5.
    1. 3 washes throughout the day is sufficient. Each at least 1hr, but exact timing isn’t important.
  7. Optional stopping point: After washing in Buffer 4, plugs can be transferred to 5mL of Buffer 5 and stored at 4C for up to 1 year. If you do not need to store plugs, proceed to step 9.
  8. If plugs were stored in Buffer 5, wash twice for 1+ hour with 15mL diluted Buffer 4 before proceeding to the next step.
  9. Put 1mL of Buffer 7 into a 2mL Eppendorf tube and transfer plug using a spatula. Make sure plug is completely immersed.
  10. Incubate at 68C for 20min.
    1. At this point, the DNA is unprotected from mechanical shearing so avoid shaking, vibration, etc. If using a water bath, use aluminum block for tubes to protect them from moving around.
  11. Transfer the tubes to 42C carefully and incubated for 10min.
    1. Do not leave a room temp for long so the agarose does not resolidify.
  12. Add 1.5uL Component 7 ( to each tube
    1. Do not mix the solution. Let the Component 7 diffuse spontaneously.
  13. Incubate overnight at 42C.

Day 3

  1. Add 1.2mL Buffer 7 to a Combireservoir (1 reservoir per sample)
  2. Gently pour the DNA solution from 42C into the reservoir
    1. Do not pipette or jostle/flick the tube – the DNA will shear. It is fine to have some solution remaining in the tube as long as the majority makes it into the reservoir.
  3. Use the Molecular Combing System to comb the DNA onto coverslips.
    1. Place combed coverslips in a 6 well dish – both sides have DNA on them but only one will be stained
    2. If possible, try to keep the coverslip oriented so you know the direction of the DNA fibers. It is much easier to image when they are horizontal rather than vertical
  4. Dehydrate combed coverslips in an incubator at 65C for 2 hours
    1. Store at -20C after or go directly to staining
  5. Denature combed coverslips in 0.5M NaOH + 1M NaCl solution for 8min at room temperature
    1. When adding liquid, use a needle to hold the coverslip down to prevent it from floating up. The coverslips are very hydrophobic until after step 7.
    2. No longer than 8 min.
    3. 1g NaOH pellets + 2.93g NaCl in 50mL water
  6. Rinse 3x in PBS
  7. Dehydrate the coverslips in 70% EtOH for 5 min
  8. Dehydrate the coverslips in 90% EtOH for 5 min
  9. Dehydrate the coverslips in 100% EtOH for 5 min
  10. Let dry at room temp
    1. Make sure to use a needle to flip the coverslip up at this point. Otherwise there is a chance it will become stuck to the bottom of the dish
  11. Incubate 1 hour in 10% goat serum/ PBSTw (0.1% Triton in PBS).
    1. Sterile filter serum, reduces background staining
    2. Serum can be reused, save and add sodium azide
  12. Incubate 1 hour in rat monoclonal anti-BrdU (anti-CldU, 1:100), mouse anti-BrdU (anti-IdU 1:100), and rabbit anti-ssDNA (1:20) diluted in 10% goat serum/PBSTw.
    1. Add 100 µL antibody to parafilm and invert coverslip on top, make sure no air bubbles are present
    2. All incubations are done in the dark, easiest to just stick in a drawer
    3. ssDNA antibody might be better at a 1:10 or 1:5
  13. Rinse 3x in PBS
  14. Incubate 30 minutes with secondary antibodies in dark. Goat anti-rat IgG Alexa Fluor 594, Goat anti-mouse Alexa Fluor 488, and Goat anti-rabbit Alexa Fluor 405 (all 1:350) in 10% goat serum/PBSTw. 100 µL per slide inverted onto parafilm.
  15. Rinse 3x in PBS
  16. Air dry in the dark
  17. Mount each with ~30 µL Prolong Gold with no Dapi
    1. Minimize air bubbles
  18. Let dry overnight in the dark and your slides are ready
    1. Can add nail polish to corners for immediate viewing
    2. After slides have dried overnight at room temperature, place them in a slide box in 4º refrigerator to preserve signal until ready to view. Recommended to view as soon as possible, or within one week.
  1. After secondary antibodies are added to the slides, protect the slides from light as much as possible.

Day 4

- Look for fibers under the red channel (IdU is green, CldU is red) to minimize photobleaching

- Fibers will be in the same plane as background specks. Go slow, fibers have a very small field of focus and are easy to pass by.

- Take a minimum of 20 pictures per condition, 5 from each quadrant of the coverslip.