Protocol: Chromatin Immunoprecipitation (ChIP) Assay		- MCPROTO407 • Product Catalogue

Part A. Optimization of DNA Shearing

Establish optimal conditions required for shearing cross-linked DNA to 200-1000 base pairs in length by following steps 1- 9 below. Vary the power setting and/or the number of 10-second pulses during sonication of the samples. Be sure to keep the sample on ice at all times (the sonication generates heat which will denature the DNA). Check the size of sonicated DNA by gel electrophoresis after reversion of cross-links. Our experience shows DNA is sheared to the appropriate length with 3-4 sets of 10-second pulses using a Cole Palmer, High Intensity Ultrasonic Processor/Sonicator, 50-watt model equipped with a 2mm tip and set to 30% of maximum power.

Alternatively; Sonication of the suspension for 30 seconds, four times with a Sonifier 450 sonicator (Branson; output 3.0, duty cycle 30%) has on ice can be used. There should be at least 1minute interval on ice between each pulse in order to cool the sample.

Note: foaming of the solution during sonication results in unequal shearing of DNA samples. To prevent foaming, keep the tip end of the sonicator near the bottom of the sample tubes, but not touching. Placing the tip end near the surface induces foaming.

Once sonication conditions have been optimized, keep cell number consistent for subsequent experiments. The protocol below for the optimization of DNA Shearing is for one Chip assay
(~1 x 10⁶ cells per condition).

Note: Steps 3 - 7 should be done on ice.

1. Stimulate or treat 1 x 10⁶ cells on a 10cm dish as appropriate. (Cells should be treated under conditions for which transcriptional activation of the gene of interest has been demonstrated). Include one extra dish (1 X 10⁶ cells) to be used solely for estimation of cell number.

2. Cross link histones to DNA by adding formaldehyde directly to culture medium to a final concentration of 1% and incubate for 10 minutes at 37ºC. (For example, add 270μl 37% formaldehyde into 10ml of growth medium on plate).

3. Aspirate medium, removing as much medium as possible. Wash cells twice using ice cold PBS containing protease inhibitors (1mM phenylmethylsulfonyl fluoride (PMSF), 1ìg/ml aprotinin and μg/ml pepstatin A). Note: Add protease inhibitors to PBS just prior to use. PMSF has a half-life of approximately 30 minutes in aqueous solutions.

4. Scrape cells into conical tube.

5. Pellet cells for 4 minutes at 2000 rpm at 4ºC. Warm SDS Lysis Buffer (Catalog # 20-163) to room temperature to dissolve precipitated SDS and add protease inhibitors (inhibitors: 1mM PMSF, 1μg/ml aprotinin and 1ìg /ml pepstatin A).

6. Resuspend cell pellet in 200μl of SDS Lysis Buffer (Catalog # 20-163) and incubate for 10 minutes on ice. Note: The 200μl of SDS Lysis Buffer is per 1 X 10⁶ cells; if more cells are used, the resuspended cell pellet should be divided into 200ìl aliquots so that each 200μl aliquot contains ~1 X 10⁶ cells .

7. Sonicate lysate to shear DNA to lengths between 200 and 1000 basepairs being sure to keep samples ice cold (Note: Once sonication conditions have been optimized following steps 1 to 9, proceed to Part B, step 1 below).

8. Add 8μl 5M NaCl (Catalog # 20-159) and reverse crosslinks at 65ºC for 4 hours.

9. Recover DNA by phenol/chloroform extraction and run sample to see the sizes of the sheared DNA (example 5ìl , 10μl and 20ìl samples) in a 2% agarose gel. Visualize shearing efficiency by staining with ethidium bromide.

Part B. Experimental Protocol

If sonication conditions have been optimized (Part A), complete steps 1 through 7 and continue with the protocol below. For a negative/background control, prepare a sample to use as a no-antibody immunoprecipitation control in step 5 below. Additionally, transcriptionally unactivated DNA samples should be prepared as controls for PCR in section II.

1. Centrifuge samples (part A, step 7) for 10 minutes at 13,000 rpm at 4 ° C, and add 200μl of the sonicated cell supernatant to a new 2ml-microcentrifuge tube. Discard pellet.

2. Dilute the sonicated cell supernatant 10 fold in ChIP Dilution Buffer (Catalog # 20-153), adding protease inhibitors as above. This is done by adding 1800ìl ChIP Dilution Buffer to the 200μl sonicated cell supernatant for a final volume of 2ml in each immunoprecipitation condition.

Note: If proceeding to PCRa portion of the diluted cell supernatant 1% (~20μl ) can be kept to quantitate the amount of DNA present in different samples at the PCR protocol, Part B, section II, step 6. This sample is considered to be your input/starting material material and needs to have the Histone-DNA crosslinks reversed by heating at 65°C for 4 hours (see section II, step 3.).

3. To reduce nonspecific background, pre-clear the 2ml diluted cell supernatant with 80μl of Salmon Sperm DNA/Protein A Agarose-50% Slurry (Catalog # 16-157C) for 30 minutes at 4ºC with agitation. Increasing the incubation time to 1 hour at 4ºC, may further reduce the nonspecific binding. This step removes proteins, which interact nonspecifically with the agarose.

4. Pellet agarose by brief centrifugation and collect the supernatant fraction.

5. Add the immunoprecipitating antibody (the amount will vary per antibody) to the 2ml supernatant fraction and incubate overnight at 4ºC with rotation. For a negative control, perform a no-antibody immunoprecipitation by incubating the supernatant fraction with 60μl of Salmon Sperm DNA/Protein A Agarose- 50% Slurry (Catalog # 16-157C) for one hour at 4ºC with rotation and proceed to step 7.

6. Add 60μl of Salmon Sperm DNA/Protein A Agarose Slurry (Catalog # 16-157C) for one hour at 4ºC with rotation to collect the antibody/histone complex.

7. Pellet agarose by gentle centrifugation (700 to 1000 rpm at 4ºC, ~1min). Carefully remove the supernatant that contains unbound, non-specific DNA. Wash the protein A agarose/antibody/histone complex for 3-5 minutes on a rotating platform with 1ml of each of the buffers listed in the order as given below:

a. Low Salt Immune Complex Wash Buffer (Catalog # 20-154), one wash
b. High Salt Immune Complex Wash Buffer (Catalog # 20-155), one wash
c. LiCl Immune Complex Wash Buffer (Catalog # 20-156), one wash
d. 1X TE (Catalog # 20-157), two washes

Section I. Immunoprecipitation/Immunoblot protocol to detect histone.

1. Following washing of the beads in part B, step 7, immunoprecipitated histones can be analyzed by immunoblot analysis. Add 25ìl of 1X Laemmli buffer per sample and boil for 10 minutes. Load 20μl per lane and perform immunoblot procedure as described per appropriate antibody.

Section II. PCR protocol to amplify DNA that is bound to the immunoprecipitated histone.

1. Freshly prepare elution buffer (1%SDS, 0.1M NaHCO 3).

2. Elute the histone complex from the antibody by adding 250ìl elution buffer to the pelleted protein A agarose/antibody/histone complex from step 7d above. Vortex briefly to mix and incubate at room temperature for 15 minutes with rotation. Spin down agarose, and carefully transfer the supernatant fraction (eluate) to another tube and repeat elution. Combine eluates (total volume = ~500μl).

3. Add 20μl 5M NaCl (Catalog # 20-159) to the combined eluates (500μl ) and reverse histone-DNA crosslinks by heating at 65ºC for 4 hours. At this step the sample can be stored and -20°C and the protocol continued the next day.

Note: Include the input/starting material (the sample saved from Part B, step 2, which has had the Histone-DNA crosslinks reversed)) as well as a transcriptionally-unactivated DNA sample as negative and background controls for the PCR reaction. Previously, a 5ìl sample has been used in a nested PCR reaction. However, the amount of sample used per reaction must be determined empirically (e.g., titrate the sample at this step by using 1, 2, 5, or 10ìl per PCR reaction) . If PCR results are poor, complete steps 4, 5 and 6 below to purify the DNA sample.

Note: Handle the samples carefully; some DNA may be lost during the purification steps.

4. Add 10μl of 0.5M EDTA (Catalog # 20-158), 20μl 1M Tris-HCl, pH 6.5 (Catalog # 20-160) and 2μl of 10mg/ml Proteinase K to the combined eluates and incubate for one hour at 45ºC.

5. Recover DNA by phenol/chloroform extraction and ethanol precipitation. Addition of an inert carrier, such as 20μg glycogen or yeast tRNA, helps visualize the DNA pellet. Wash pellets with 70% ethanol and air dry.

6. Resuspend pellets in an appropriate buffer for PCR or slot-blot reactions. PCR or slot-blot conditions must be determined empirically. The following are conditions that have been successfully used at Upstate:

PCR conditions for the Fos promoter in 3T3 cells

First round PCR:

• GGCGAGCTGGTTCCCGTCAATCC-5’ primer
• TGCAGTCGCGGTTGGAGTAGTAGG-3’ primer
• 5 microliters DNA sample from post 65C reverse cross-linking
• 2 microliters input DNA collected at step #6
• No DNA control
• Buffer conditions: 2.5 units Taq DNA Polymerase (add last), 10mM Tris HCl, 50mM KCl, 1.5mM MgCl2, 200uM dNTP’s, 1.0uM forward and reverse primers, mix in final volume of 100ìl.

Step 1: 94C for 3 min
Step 2: 94C for 1 min.
Step 3: 57C for 2 min.
Step 4: 72C for 3 min.
Step 5: go to step 2, 19 times
Step 6: 72C for 5 min.
Step 7: OC for 0 min.

Second round PCR:

• TCCACGGCCGGTCCCTGTTGTTCT-5’ primer
• GCGGGCGCTCTGTCGTcAACTCTA-3’ primer
• 2 microliters from first round
• Buffer conditions: 2.5 units Taq DNA Polymerase (add last), 10mM Tris HCl, 50mM KCl, 1.5mM MgCl2, 200uM dNTP’s, 1.0uM forward and reverse primers, mix in final volume of 100μl.

Step 1: 94C for 3 min
Step 2: 94C for 1 min.
Step 3: 60C for 2 min.
Step 4: 72C for 3 min.
Step 5: go to step 2, 19 times
Step 6: 72C for 5 min.
Step 7: OC for 0 min.

Run 2 ^nd round PCR product on a 1.4% agarose gel. 30μl of product + 3μl of dye.