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 10cm 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 8mL 1% Formaldehyde (in PBS) and incubating 10 min at room temperature.
  3. Wash 3 times with cold PBS.
  4. Scrape cells into 1mL PBS (+protease/phosphatase inhibitors) and pellet at 3000rpm for 3 min at 4C.
  5. Prepare crude nuclei: Resuspend cells in 1mL 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 4C.
  6. Resuspend nuclear pellet in 250 ul 1% SDS lysis buffer (+inhibitors). Incubate on ice 10 min.
  7. Shear DNA using Menon Lab cup horn sonicator (set at 4.5…see Tracy Cui in 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 4C.
  8. After sonication, run 20ul of sample in 1.5% DNA agarose gel to verify that sonication has resulted in 200-1000bp fragments of DNA.
  9. Quantify protein in supernatant by standard protein assay (biorad reagent) and use 100ug protein per IP. Bring volume of each IP up to 100ul with 1% SDS lysis buffer. Dilute 1:10 by adding 900ul of ChIP dilution buffer.
  10. Save 10ul for 1% input (store at –20 until time to process).
  11. To reduce nonspecific background, pre-clear each IP sample with 60ul salmon sperm DNA/BSA/protein A agarose beads (homemade or from upstate biotechnology), 10ul of rabbit pre-immune serum (Pierce cat# 31884) and 5ug sheared herring sperm DNA. Incubate for 1-2 hours rotating at 4C. Pellet agarose by brief centrifugation at 1000rpm for 1 min at 4C and keep the supernatant, discard the pellet.
  12. Add the immunoprecipitating antibody (amount will vary per antibody…1-2ug) to the supernatant fraction and incubate overnight at 4C with rotation. For a negative control, use a non-specific antibody for immunoprecipitation.

 

Day 2:

  1. Add 40ul DNA/BSA/Protein A Agarose and 10ug sheared herring sperm DNA to samples and rotate another 1-2 hours.
  2. Pellet agarose by gentle centrifugation (700 rpm for 1 min at 4C). Carefully remove and discard the supernatant the contains unbound, non-specific DNA. Washt he protein A agarose/antibody/protein complex for 3-5 minutes rotating at 4C 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)

 

  1. Freshly prepare elution buffer (1% SDS, 0.1M NaHCO3). Recipe to make 10mL: (84mg NaHCO3+1mL10% SDS+9mL H2O).
  2. Elute from beads by adding 110ul 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 110ul elution buffer to the beads and repeat. Combine elutions into one tube so that total volume is around 200-220 ul.
  3. Add 10ul of 5M NaCl and 1ul Rnase (10mg/ml) to the elutions and reverse protein/histone-DNA crosslinking by heating at 65C for 4 hours (or overnight). At this step the sample can be stored at –20C until next step. Remember the 1% inputs that you’ve stored at -20? Add 200ul elution buffer, 10ul 5M NaCl and 1ul Rnase (10mg/ml) to each 10ul input and incubate at 65C for 4 hours (or overnight) along with ChIP samples.

 

Day 3:

  1. Digest proteins in sample by adding 10ul of 0.5M EDTA, 20ul 1M Tris-HCl (pH 6.5) and 2ul 10mg/ml Proteinase K for one hour at 45C.
  2. Recover DNA using Qiagen PCR purification kit. Final elution is in 50 ul EB. Store sample at –20C.
  3. Perform PCR analysis (try 1 to 2 ul of sample for each PCR in a 25ul volume reaction).

 

 

Note: To verify effective sonication (200-1000bp), run out 20ul of sonicated lysate in 1.5% gel. For more accuracy (especially when troubleshooting sonication conditions), reverse crosslinks by adding 5ul 5M NaCl to 100ul of the lysate and incubating at 65C for 4 hours (or overnight). Then run out on gel to verify length of fragments. Expect to see a smear of DNA from 200-1000bp (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 10ul Western lysis buffer and 10ul 4X SDS LB. Heat to 95C 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, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl

High Salt Immune Complex Wash Buffer: 0.1% SDS, 1%Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl

LiCl Immune Complex Wash Buffer: 0.25M LiCl,1% NP40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl, pH 8.1.

 

 

 

Supplemental Protocol: To make herring (or salmon) sperm DNA/protein A agarose

(Courtesy of Upstate Biotechnology)…keep at 4C

 

 

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 Ķ

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 4oC.

5. Aliquot and store at 4oC.