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iPOND 1.0

iPOND Methodology

iPOND protocol_BMS download

Materials:

REAGENTS

  • EdU (5-ethynyl-2’-deoxyuridine) (Invitrogen, cat. no E10187)
  • 37% (wt/vol) formaldehyde solution (Sigma, cat. no F1635)
  • 10X PBS pH 7.2 (Gibco, cat. no 70013)
  • Glycine (Fisher, cat no BP 381)
  • Cell scraper
  • Triton X100
  • Bovine serum albumin
  • DMSO
  • Invitrogen’s Click-it Cell Reaction Buffer Kit (Invitrogen, cat. no C10269)
    • Click-iT Cell Reaction Buffer (10X solution containing Tris-buffered saline) substitute PBS
    • Copper (II) sulfate (CuSO4) (100mM aqueous solution)
    • Click-iT Cell Buffer Additive substitute Na ascorbate
  • Biotin azide (Invitrogen, cat. no B10184)
  • SDS
  • Tris
  • Aprotinin (Sigma, cat. no A6279)
  • Leupeptin (Sigma, cat. no L2884)
  • 90 micron nylon mesh
  • Streptavidin agarose (Novagen, cat. no 69203-3)
  • NaCl

EQUIPMENT

  • Microtip sonicator for cell lysis and chromatin fragmentation (Misonix 4000 or Fisher Scientific Sonic Dismembrator Model 500)
  • Rotating platform for biotin captures o/n @ 4C

REAGENT SETUP

  • EdU
    • Dissolve EdU in DMSO for a final stock concentration of 10mM. Aliquot and store at -20C in the dark. Stable for up to 1 year.
    • Before use, warm up at 37C in dark for a couple of minutes. To pulse cells, pipet 1:1000 of EdU directly into media for a final concentration of 10uM.
  • 1% formaldehyde/PBS (prepared freshly)
    • Dilute 37% formaldehyde 1:37 with PBS. Keep at RT until cells fixation step.
  • 1.25M Glycine
    • Prepare 1.25M glycine stock. Store at RT. Use at 1:10 dilution for final concentation of 0.125M glycine.
  • Permeabilization buffer
    • Make stock 20% Triton-X and keep at RT.
    • Dilute to 0.25% Triton-X in PBS and keep at 4C.
  • 0.5% BSA/PBS wash buffer
    • Prepare 0.5% BSA in PBS. Filter and store at 4C.
  • Click reaction cocktail (prepared freshly)
    • Dissolve biotin-azide in DMSO to achieve a final concentration of 1mM. Aliquot and store at -20C.
    • Prepare stock 100mM CuSO4 in MQ H2O. Store at RT.
    • Prepare 20mg/ml of (+) sodium L-ascorbate (reducing agent) in H2O and store on ice until needed. Prepare freshly every time and limit exposure to air.
    • Make 1X PBS from 10X PBS stock and store at RT.
  • Lysis buffer
    • Prepare 1% SDS in 50mM Tris, pH 8.0. Store at RT.
    • Prior to use, add protease inhibitors aprotinin and leupeptin to final concentration of 1ug/ml.
  • Salt wash
    • Prepare 5M NaCl and store at RT. To wash iPOND captures, dilute to 1M NaCl.
  • Elution buffer
    • Prepare 2X SB and store at -20C. Add DTT freshly.

Procedure:

DAY 1—

  1. Expand ___ of 150mm^2 plates of 293T cells into ___ plates.
  2. Tomorrow, 3 plates will be collected per each of the experimental samples.

*CRITICAL STEP

  • Experiment works best when cells have fresh media and are expanded one day before experiment.

DAY 2—

Count cells

  1. Count one 150mm^2 plate of 293T cells.
  2. This dish contains equal density of cells as dishes that will be used for this experiment.

*CRITICAL STEP

  • Experiment works best when cell confluence is between 4-6 x 10^7 cells / dish
  • Cells must be in log phase of growth and cannot be overgrown. EdU incorporation is not maximal unless this critical parameter is met.

EdU Pulse

  1. Write down samples below:

Example:
1 = 10 min EdU, no biotin-N3 (negative control)
2 = 10 min EdU, + biotin-N3

  1. Plan out times to pulse, fix, quench, collect and wash samples PRIOR to beginning pulsing cells.

*CRITICAL STEP

  • Stagger samples to ensure that each sample is treated equally through processing steps.
  • Each samples requires ca. 5 minutes of processing time to collect and 20 minutes of washes.
  • To pulse cells with EdU, remove 3 plates from incubator and take into culture hood.
  • Pipet 23ul of 10mM EdU stock into 23ml of media containing cells to achieve final [EdU] = 10uM.

*CRITICAL STEP

  • It is important to perform this step as fast as possible to prevent pH and temperature changes in the media that can affect replication rates.
  • Place plates back into incubator to pulse with EdU for 10 minutes.
  • If not performing chases into thymidine or drug treatments, skip to step 14.
  • To chase samples, remove plates from incubator and decant media.
  • Carefully wash cells with 4-5ml of chase media.
  • Decant.
  • Carefully add back 20ml of media containing 10uM thymidine or desired concentration of DNA damaging drug.
  • Chase samples for desired amount of time.

Collect and fix cells with HCHO (formaldehyde)

  1. After EdU pulse and/or chase, decant media.
  2. Immediately fix cells on plate by adding 10ml of 1% formaldehyde (HCHO) in PBS.
  3. Incubate cells 20 mins @ RT.

*CRITICAL STEP

  • Stagger samples to ensure that each sample is treated equally through processing steps.
  • Fix one sample at a time.
  • Quench cross-linking by adding 1ml of 1.25M glycine.
  • As soon as glycine has been added, begin to collect sample by scraping with cell scraper.
  • Collect cells in 50ml conical.
  • Note: It is not necessary to wash plates, unless majority of cells were not scrapped off.
  • Note the volume. This is same volume to be used for PBS washes.
  • Centrifuge tubes 5mins @ 4C @ 2,000rpm.
  • Decant supernatant.
  • Wash pellets 3X with PBS and spin down 5 mins @ 2,000rpm @ 4C.
  • Note: PBS wash volume is same as fixation volume noted above.
  • After last wash, decant PBS.

Stopping point—samples can be stored at -80C.

Permeabilize cells

  1. Resuspend cells in 0.25% TritonX/PBS at concentration of 1 x 10^7 cells/ 1mL.
  2. Incubate cells @ RT 30 mins (during incubation, prepare Click Rxn, see step 32).
  3. Spin down 5 mins @ 2,000rpm @ 4C
  4. Carefully decant supernatant.
  5. Wash cells 1X with cold 0.5% BSA/PBS.

*CRITICAL STEP

  • BSA prevents cell pellet from detaching from wall of 50mL conical.
  • Loose pellet will lead to loss of cells in this step.
  • Spin down 5 mins @ 2,000rpm @ 4C and decant supernatant.
  • Wash cells 1X with PBS.
  • Spin down 5 mins @ 2,000rpm @ 4C, decant supernatant and place pellets on ice while finishing Click rxn cocktail preparation.

Click Reaction

Preparation and Click reaction cocktail calculations

  1. During cell permeabilization, take out necessary reagents for click reaction.
  2. Warm up an aliquot of stock biotin-azide by placing on 37C heat block.

*CRITICAL STEP

  • If using photocleavable biotin-azide, keep reagent protected from light and prepare Click reaction cocktail in the dark.
  • To calculate click reaction cocktail volumes:
  • Note that 1 reaction (1x rxn) is sufficient to biotin tag 1 x 10^7 cells.
  • Each 1x rxn contains 500ul of added reagents.
  • Use table below to calculate amounts of each reagent needed per sample.
  • Note that 2 click reaction cocktails will need preparation: 1 for the negative control, which contains DMSO, and 1 for the experimental samples, which contains biotin-azide.
  • An example is given for a sample size of 10 x 10^7 cells.

NEGATIVE CONTROL (DMSO, no Click)

EXPERIMENTAL SAMPLES (Biotin-azide, Click rxn)

  1. After calculations are complete, add each reagent to 50ml conical tube on ice in the order listed in the tables above.
  2. Resuspend pellets by vortexing.
  3. Rotate reactions @ RT for 1hr.
  4. Spin down Clicked samples 5 mins @ 2,000 rpm @ 4C and decant supernatant.
  5. Wash cells 1X with cold 0.5% BSA/PBS using same volume as used in click reaction for one sample.

*CRITICAL STEP

  • BSA prevents cell pellet from detaching from wall of 50mL conical.
  • Loose pellet will lead to loss of cells in this step.
  • Spin down 5 mins @ 2,000rpm @ 4C and decant supernatant.
  • Wash cells 1X with PBS.
  • Decant PBS and invert tubes on paper towel to remove all PBS.

Stopping point—samples can be stored at -80C.

Lysis and sonication

  1. Prepare Lysis Buffer by adding aprotinin and leupeptin before use and place on ice.
  2. Resuspend samples at a concentration of 8.8 x 10^7 cells per 600ul lysis buffer and place into 1.5ml Eppendorf tubes on ice.

*CRITICAL STEP

  • Make certain that all cells have been washed off of 50ml conical tube.
  • During sonication, keep samples being sonicated on ice slurry to prevent overheating.
  • Sonicate cells using microtip sonicator and the following settings:
    • Pulse: 20 seconds constant pulse, 40 seconds pause
    • Power: 13-16 Watts
    • Pulse: 1 X for every 200ul of cell lysate
    • Total pulse time: 4-5 minutes per sample

*CRITICAL STEP

  • Lysate should appear clear/opaque after sonication and not cloudy.
  • Cloudiness is an indicator of improper ratio of SDS to protein in lysate.
  • Centrifuge samples 10 mins @ max speed RT in table-top centrifuge.
    • Sample will be warm to the touch after spin.
  • Filter supernatant through a 90 micron nylon mesh into 15ml conical tube. Place on ice.
  • Note the LYSATE VOLUME = _________________
  • Dilute lysate 1:1 (v/v) with cold PBS containing 1ug/ml of aprotinin and leupeptin.

*CRITICAL STEP

  • Samples have been diluted to contain 0.5% SDS, 25mM Tris because biotin capture is inefficient in lysates containing 1% SDS.
  • Note the FINAL CAPTURE VOLUME = _________________
  • Remove 15ul of lysate at this point and place on ice. Add 2X SDS buffer in 1:1 (v/v) and freeze at -80C. This represents the Input for each capture.

Biotin capture

  1. To capture biotin-tagged nascent DNA, each sample is incubated with streptavidin agarose beads at a concentration of 100ul bead slurry (50ul packed volume) per 1 x 10^8 cells.
  2. Wash beads for all samples together by centrifuging bead slurry @ 1800g for 1min @ RT.
  3. Slowly and carefully aspirate storage buffer off of beads using a gel loading tip.
  4. Wash beads 2 X with 1:1 (v/v) of Lysis Buffer.
  5. Wash beads 1 X with 1:1 (v/v) of PBS containing aprotinin and leupeptin.
  6. Resuspend beads 1:1 (v/v) in PBS containing protease inhibitors.
  7. Add equal volume of beads to each sample using pipet tip that is cut at the end.
  8. Rotate biotin captures in cold room 16-20 hrs.

DAY 3—

  1. Take down captures after ________hrs of capture.
  2. Spin down beads with captured DNA and associated proteins @ RT 3 mins 1800g.
  3. Very slowly and carefully aspirate most of supernatant.
    • Note: Supernatant should be light blue/clear.
  4. Wash beads with 1ml of cold Lysis Buffer (no additives needed).
  5. Rotate beads on rotating platform @ RT for 5 mins.
  6. Spin @ RT 1min 1800g.
  7. Wash 1x with 1ml of 1M NaCl.
  8. Rotate and pellet beads as in 66-67.
  9. Repeat Lysis Buffer washes 2X.
  10. After last wash, aspirate off all supernatant.
  11. To elute proteins bound to nascent DNA, add 2X SB to packed beads 1:1 (v/v of packed beads; example: 90ul 2X SB : 90ul packed beads).
  12. Very lightly flick beads to mix.
  13. Quick spin to pellet beads.
  14. To finish protein elution, boil captures and inputs for 25mins @ 95C.
  15. Quick spin.
  16. Calculate volume of input needed to load per well to achieve 0.1% input.
  17. Pour, load, run and transfer gels.

DAY 4—

  1. Finish Western blotting.