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Chromatin Immunoprecipation (ChIP) assay for 3T3-L1 preadipocytes

(Jennifer Kennell, 4/05)

Note: conditions will have to be worked out for each cell type (preads vs. ads, ST2s, etc). Also the protocol here has not been perfected for 3T3-L1 preadipocytes yet since I still get non-specific pulling down of DNA. More troubleshooting will be required. But use this as a starting point
 
Grow 3T3-L1 preadipocytes to two days post-confluence in 10 cm plates and treat according to experimental design. Each plate will give around 4-6 different assays, the exact amount will be determined after sonication.
 
Day 1:
  1. Wash preadipocytes once with room temp PBS.
  2. Crosslink by adding 8 mL 1% Formaldehyde (in PBS) and incubating 10 min at room temperature.
  3. Wash 3 times with cold PBS.
  4. Scrape cells into 1 mL PBS (+protease/phosphatase inhibitors) and pellet at 3000 rpm for 3 min at 4 C.
  5. Prepare crude nuclei: Resuspend cells in 1 mL hypotonic buffer (+inhibitors) and incubate on ice 10 min. Dounce homogenize with 20 strokes and briefly spin down nuclei at 13,200 rpm for 30 sec at 4 C.
  6. Resuspend nuclear pellet in 250 µL 1% SDS lysis buffer (+inhibitors). Incubate on ice 10 min.
  7. Shear DNA using Menon Lab cup horn sonicator (set at 4.5, see Schwartz lab about how to use). Use 7X15 sec bursts with at least one minute of incubation on ice between bursts. After sonication, spin down cellular debri by centrifuging at 13,200 rpm for 5 min at 4 C.
  8. After sonication, run 20 µL of sample in 1.5% DNA agarose gel to verify that sonication has resulted in 200-1000 bp fragments of DNA.
  9. Quantify protein in supernatant by standard protein assay (biorad reagent) and use 100 µg protein per IP. Bring volume of each IP up to 100 µL with 1% SDS lysis buffer. Dilute 1:10 by adding 900 µL of ChIP dilution buffer.
  10. Save 10 µL for 1% input (store at –20 until time to process).
  11. To reduce nonspecific background, pre-clear each IP sample with 60 µL salmon sperm DNA/BSA/protein A agarose beads (homemade or from upstate biotechnology), 10 µL of rabbit pre-immune serum (Pierce cat# 31884) and 5 µg sheared herring sperm DNA. Incubate for 1-2 hours rotating at 4 C. Pellet agarose by brief centrifugation at 1000 rpm for 1 min at 4 C and keep the supernatant, discard the pellet.
  12. Add the immunoprecipitating antibody (amount will vary per antibody, 1-2 µg) to the supernatant fraction and incubate overnight at 4 C with rotation. For a negative control, use a non-specific antibody for immunoprecipitation.
 
Day 2:
  1. Add 40 µL DNA/BSA/Protein A Agarose and 10 µg sheared herring sperm DNA to samples and rotate another 1-2 hours.
  2. Pellet agarose by gentle centrifugation (700 rpm for 1 min at 4 C). Carefully remove and discard the supernatant the contains unbound, non-specific DNA. Wash the protein A agarose/antibody/protein complex for 3-5 minutes rotating at 4 C with each of the buffers listed in the order as given below. After final TE wash, carefully remove all liquid using a gel loading pipette tip.
    a)     Low Salt Immune Complex Wash Buffer (1 X 1mL)
    b)    High Salt Immune Complex Wash Buffer (1 X 1mL)
    c)     LiCl Immune Complex Wash Buffer (1 X 1mL)
    d)    1X TE (2 X 1mL)
  3. Freshly prepare elution buffer (1% SDS, 0.1 M NaHCO3). Recipe to make 10 mL: (84 mg NaHCO3+1 mL 10% SDS + 9 mL H2O).
  4. Elute from beads by adding 110 µL elution buffer, vortex and rotate at room temp for 15 minutes. Carefully transfer supernatant to new tube (make sure no beads carry-over!). Add another 110 µL elution buffer to the beads and repeat. Combine elutions into one tube so that total volume is around 200-220 µL
  5. Add 10 µL of 5 M NaCl and 1 µL RNAse (10 mg/ml) to the elutions and reverse protein/histone-DNA crosslinking by heating at 65 C for 4 hours (or overnight). At this step the sample can be stored at –20 C until next step. Remember the 1% inputs that you've stored at -20? Add 200 µL elution buffer, 10 µL 5 M NaCl and 1µL Rnase (10 mg/ml) to each 10 µL input and incubate at 65 C for 4 hours (or overnight) along with ChIP samples.

Day 3:
  1. Digest proteins in sample by adding 10 µL of 0.5 M EDTA, 20 µL 1 M Tris-HCl (pH 6.5) and 2 µL 10 mg/mL Proteinase K for one hour at 45 C.
  2. Recover DNA using Qiagen PCR purification kit. Final elution is in 50 µL EB. Store sample at –20 C.
  3. Perform PCR analysis (try 1 to 2 µL of sample for each PCR in a 25 µL volume reaction).
Note: To verify effective sonication (200-1000 bp), run out 20 µL of sonicated lysate in 1.5% gel. For more accuracy (especially when troubleshooting sonication conditions), reverse crosslinks by adding 5 µL 5 M NaCl to 100 µL of the lysate and incubating at 65 C for 4 hours (or overnight). Then run out on gel to verify length of fragments. Expect to see a smear of DNA from 200-1000 bp (not distinct fragments).
 
Note: To verify that the immunoprecipitation of protein is working, prepare ChIP samples (with or without IP antibody) and after step 14 (washing of beads) add 10 µL Western lysis buffer and 10 µL 4X SDS LB. Heat to 95 C for 10 minutes to reverse all crosslinking and perform a western.



Recipes: no DTT in any of the recipes!!!!!!
Hypotonic Lysis Buffer: 20mM Tris-HCl, pH 7.5, 10mM NaCl, 3mM MgCl2
SDS Lysis Buffer: 1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1. (Store at RT)
ChIP Dilution Buffer: 0.01% SDS, 1.1% Triton X-100,1.2mM EDTA, 16.7mM Tris-HCl, pH 8.1, 167mM NaCl

Low Salt Immune Complex Wash Buffer: 0.1% SDS, 1%Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl
High Salt Immune Complex Wash Buffer: 0.1% SDS, 1%Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl
LiCl Immune Complex Wash Buffer: 0.25 M LiCl,1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1.
 
  

Supplemental Protocol: To make herring (or salmon) sperm DNA/protein A agarose:
(Courtesy of Upstate Biotechnology), keep at 4 C
  1. Make 100 ml of sterile TE (10 mM Tris, pH 8/1 mM EDTA, pH 8).
  2. Combine: 50 mg BSA (the one used for diluting antibodies)
    • 0.5 ml Sodium Azide (from a 5% stock solution)
    • Bring up to 47.5 ml with TE and filter sterize using a 0.2 µm filter)
  3. Wash 20 ml of protein A beads (50% slurry catalog 16-125, Upstate Biotechnologies)
    • twice using 15 ml of sterile TE.
  4. Combine: 10 ml washed protein A packed beads
    • 4.0 mg herring (or salmon) sperm DNA (sonicated)
    • bring up to 20 ml using sterile TE/BSA/sodium azide solution rock 45 minutes at 4 C.
  5. Aliquot and store at 4 C.