CHIP Protocol

Johannes (protocol is a mix between Antoine Peters lab, FMI, Basel, Switzerland and Upstate’s 17-285)

Day one: Total time: 10-11h

Preparation of solutions before starting (all in ddH2O unless otherwise stated):

TE

  • 10 mM Tris-Cl, pH 8.0
  • 1 mM EDTA

Fix solution

  • 50mM HEPES ph7.5 (=5ml of 0.5M stock in 50ml)
  • 1mM EDTA ph8 (=100ul of 0.5M stock in 50ml)
  • 0.5mM EGTA (=125ul of 200mM stock in 50ml)
  • 100mM NaCl (=1ml of 5M stock in 50ml)

Formaldehyde* solution:

  • 70% Fix Solution (see above) 30% 37%-Formaldehyde (Sigma F1635).
  • Dilute 1/10 of the above solution in sterile-filtered PBS (final formaldehyde 1%). Per sample, 5ml of 1% final formaldehyde solution are needed.

Glycine 2.5M (Sigma G7403)

  • per 5ml formaldehyde solution, 0.25ml (1/20) are needed per sample.

Washing Buffer prior to lysis

  • Cold sterile-filtered PBS containing proteinase inhibitors (Roche), 1 tablet per 10ml. Per sample, 10ml are needed.

Cell Lysis Buffer*

  • 10mM Tris-HCl ph8
  • 10mM NaCl
  • 0.2% NP-40
  • + Proteinase Inhibitors (see above)
  • + Phosphatase Inhibitors if needed

per 1ml

      • 10ul 1M b-glycerophosphate
      • 10ul 1M NaF
      • 1ul of 100mM Na3VO4
  • Per sample 5ml are needed

Nuclear Lysis Buffer*

  • 1% SDS (=2.5ml of 20% stock in 50ml)
  • 10mM EDTA ph8 (=1ml of 0.5M stock in 50ml)
  • 50mM Tris-HCl ph8 (=2.5ml of 1M stock in 50ml)
  • + Proteinase Inhibitors (see above)
  • + Phosphatase Inhibitors if needed (see above)
  • Per sample 1ml is needed

Dilution Buffer

  • 1% Triton X-100 (=5ml of 10% stock in 50ml)
  • 150mM NaCl (=1.5ml of 5M stock in 50ml)
  •  2mM EDTA ph8 (=0.2ml of 0.5M stock in 50ml)
  • 20mM Tris ph8 (=1ml of 1M stock in 50ml)
  • + Proteinase Inhibitors (see above)
  •  + Phosphatase Inhibitors if needed (see above)
  • quantity has to be determined after [ ] measurement

* prepare freshly!

Fixation and lysis of tissue

  1. Dissection of structure on ice or N2(l). Chop into smallest pieces possible. Separate pieces with foreceps.
  2. Add 5ml 1% Formaldehyde solution
  3. Incubate rotating for 15min at RT
  4. Add .25ml 2.5M Glycine to each sample (final [ ] = .125M), mix by inverting tube.
  5. Spin 1500rpm for 5min at 4°, get rid off SN
  6. Wash pellet with cold PBS (containing proteinase inh), 5ml each time
    1. Add solution
    2. Mix by shaking
    3. Spin 1500rpm for 5min at 4°, get rid off SN
  7. Repeat step 6
  8. Homogeneize sample in 2ml Cell Lysis Buffer (containing proteinase and phosphatase inh) in Dounce Homogeneizer 10 strokes on ice. Rinse pestle with additional 3ml Cell Lysis Buffer (containing proteinase and phosphatase inh)
  9. Spin 4000rpm for 5min at 4°, get rid off SN
  10. Homogeneize sample in 1000ul of Nuclear Lysis Buffer (containing proteinase and phosphatase inh) with P1000 several strokes on ice
  11. Incubate 10min on ice (SDS may precipitate, if so, put back at RT, wait and shake gently

Sonication: 10 x 15s at 25% power (setting “5”) (Branson Digital Sonifier) in groups of 3

Store at 4°; at -80° for long-term storage

Post-Sonication for shearing control

  1. Mix
    1. 80ul Nuclear Lysis Buffer (NO inhibitors)
    2. 20ul sample
    3. 25ul RNase A (Invitrogen 12091-039, final [ ]=0.385mg/ul)
  2. Incubate 15min at 37° (heating block)
  3. Add 5ul Proteinase K (10mg/ul)
  4. Incubate 30min at 37° (heating block)
  5. Incubate 3h at 65° (heating block)
    • During that time, prepare 2% agarose gel
  6. Spin down samples
  7. Add 125ul Phenol/Chloroform mix, vtx 10s, spin 5min at FS, RT
  8. Prepare new tubes, transfer the upper phase without touching of the lower phase to the new tubes (ca. 100ul). Add 1/50 x 5M NaCl (final [ ] 0.1M (ca. 2ul).
  9. Add 2.5 volumes of ice-cold 100% EtOH (250ul roughly), mix by shaking
  10. Incubate 30min at -20°.
  11. Spin 10min at FS, 4°
  12. Take out liquid on correct side of the tube (P1000).
  13. Add (Wash) with 200ul ice-cold 70% EtOH
  14. Spin 1min at FS, 4°
  15. Repeat step 12 and take out every drop this time (P200).
  16. Speed-vac (medium setting) at least 7min; no EtOH may be left after this step
  17. Add 20ul TE Buffer, mix by flicking, brief spin
  18. Incubate 5min at RT, mix by flicking, brief spin
  19. Load on 2% agarose gel, for each sample 7 and 14ul, after adding 3-4ul of Loading Dye; run gel at low V.

During that time, measure [ ] of sonicated chromatin on nanodrop

Dilute 1:5 in Dilution Buffer (see above), total V=5ul.

IP

  1. Calculate the amount needed per IP: Use 100ug to 150ug per sample per IP; ½ of that for input control.
  2. Transfer the required amount (plus 10 spare ul) to fresh Epi tubes
  3. Spin down samples 5min, FS, RT
  4. Take away supernatent (containing the sample) and distribute into fresh Epi tube (one tube for the IP with the antibody, one tube for the input control)
  5. a) Dilute sample 1:10 in Dilution Buffer containing the corresponding inh.  (e.g. for 140ul sample add 1260ul Dilution Buffer. b) for input control, do not add Dilution Buffer.
  6. Add 2-5ul of the desired ab (including a non-specific, “mock” ab, such as IgG; this is the non-specific ab-control – if desired).
  7. Incubate samples and input control rotating O/N at 4°.
Day two: Total time 9-10h

Addition of beads: Add 60ul of Protein A Agarose/Salmon Sperm DNA (50% Slurry) from Upstate kit (product number 16-157C) and continue incubation at 4° for a min of 60min

During that time, prepare the following buffers:

Wash Buffer (per sample 3ml)

  • 0.1% SDS in Dilution buffer
  • + proteinase inhibitors

Final Wash Buffer (per sample 1ml)

  • 1% Triton X-100 (=5ml of 10% stock solution in 50ml)
  • 0.1% SDS (=250ul of 20% stock solution in 50ml)
  • 0.5M NaCl (=5ml of 5M stock solution in 50ml)
  • 2mM EDTA ph8.0 (=0.2ml of 0.5M stock solution in 50ml)
  • 20mM Tris-Cl ph8.0 (=1ml of 1M stock solution in 50ml)
  • + proteinase inhibitors

TE (per sample 2ml) (see above)

Washing of beads-samples (not for Input control)

  1. Spin beads-samples at 2.5krpm for 2min at 4°
  2. Remove SN and add 1ml Wash Buffer, mix by flicking/ inverting, incubate           10min rotating at 4°
  3. Repeat steps 1 and 2 another two times, so that beads-samples get washed a             total of 3 times in Washing Buffer
  4. Remove SN and add 1ml Final Wash Buffer, mix by flicking/ inverting,      incubate 10min rotating at 4°
  5. Spin beads-samples at 2.6krpm for 1.5min at 4°
  6. Remove SN and add 1ml TE, mix by flicking/ inverting, incubate 10min rotating     at 4°
  7. Spin beads-samples at 2.6krpm for 1.5min at 4°
  8. Repeat steps 6 and 7 one more time, for a total of 2 TE washes
  9. Remove remaining liquid carefully with P1000, P200
    1. Add 240ul 1% SDS/ TE mix (prepare during one of the washes) to the beads-samples and incubate rocking vigorously at 65° for 10min
    2. Add equal V of Nuclear Lysis Buffer and 300ul TE to input control
  10. Incubate both beads-samples and input control at least 4h (but no longer than 10h) at 65°.
  11. Store samples at -20°.
Day three: 3 hours

Reverse crosslink:

  1. Add equal V of TE to samples (not to input control)
  2. Add 10ul of Proteinase K (20mg/ml) to samples and to input control (final[ ]=0.4mg/ml), mix by flicking and incubate 1h at 45° on heating block

Extract DNA using Phenol/Chloroform/Isoamyl Alcohol (Fluka):

  1. Add equal V to samples and input control
  2. Vortex 30sec
  3. Spin at 13krpm at RT for 15min
  4. Transfer SN to new tube
  5. Add 5M NaCl to a final [ ] of 0.1 M
  6. Add 1ul Glycogen (Roche, Nr. 10 901 393 001)
  7. Add 2.5x V of ice-cold 100% EtOH (Fluka)
  8. Put at -20° for a minimum of 30min
  9. Spin at FS at 4° for 14min
  10. Take away SN
  11. Add 1ml 70% ice-cold EtOH
  12. Spin at FS at 4° for 1min
  13. Speed-vac at 30° for 10min
  14. Resuspend in 100ul ultrapure water

Dilute Input samples 1:10.

Store at -20°. Run qPCR with gene or promoter-specific primers.