A. Magnetic beads- antibody preparation (Day 1)

1. Take 50ul or 10ul of beads/ ChIP condition 

Note- volumes vary depending on the size of the ChIP. We take 50ul of beads for ~7 million cells chromatin for NFAT/POL2 ChIPs, and only 10ul of beads for ~1 million cell chromatin for H3K4me2.

2. Wash with 1.5ml blocking solution (PBS + 0.5% BSA), incubate 5 min, spin beads at 4°C for 1 min 1000xg.

3. Repeat step 2 2 times.

4. Resuspend in Nx50ul to get back the initial quantity with blocking solution.

5. Divide the volume and add antibodies (specific for every experiment)--125ul/CjhIP (beads + Ab + blocking solution).

Ex: NFAT ChIP: 600ul beads + 30ul Ab (2ug/ul) + 870 blocking solution = 12 ChIP conditions.

6. Incubate at 4°C for at least 5 hours while rotating.

7. Wash beads with 3x 1ml RIPA/lysis buffer.

8. Add 50ul beads to the diluted chromatin (prepared below).


B. Preparation of the chromatin (for more detailed protocol see "DNA fragmentation by sonication")- Day 1

1. Take pellets from freezer, keep on dry ice.

2. Add 120 ul of complete lysis buffer to 10 million cell pellets, incubate 2 min at room temp, put on ice.

3. Transfer to 0.5ml axygen tubes.

4. Perform 20 cycles of sonication: 30" on/off, high power.

5. Check sonication by gel.

6. If sonication right, pool similar conditions and add 900ul of complete RIPA buffer (ice cold) for every 120ul of lysis buffer.

7. Spin at max speed for 15 min at 4°C. 

8. Prepare tubes for ChIPs and Inputs.

9. Collect 1000ul of diluted chromatin in Input tubes; Transfer 800ul to ChIP tubes. 

Note- volumes may vary depending on the size of the ChIP. For example, we do 800ul for NFAT or POL2 ChIPs in 1.5ml axygen tubes, but only 100ul for H3K4me2 ChIPs in 0.2ml axygen tubes.

10. Add 50/10ul of beads+ab complexes in diluted chromatin. 

11. Incubate overnight at 4°C on rotating platform. Keep the rest at -20°C.


C. Washing- Day 2

1. Pre-chill one 1.5ml tube for each IP.

Transfer the volume of ChIP samples to the new pre-chilled tube.

3. Let tubes sit in magnetic device to collect the beads. Remove supernatant (save if you want it as a control later). 

4. Add 1ml (150ul for small ChIP) RIPA to each tube. Remove tubes from magnet and invert gently to resuspend the beads. Place tubes on the rotating platform for 5min at 4°C. 

5. Place tubes on magnet to collect beads, remove supernatant.

6. Repeat steps 3-5 with 1x RIPA, 1x high salt buffer, 1x LiCl, and 1x low salt buffer (TE + 50mM NaCl).

7. Resuspend in 1ml (150ul) of TE, transfer to new pre-chilled tubes (Note- may be necessary to pre-wash these tubes with RIPA buffer; vortex and aspirate).

8. Replace tubes in magnetic device to collect beads. Remove supernatant.


D. Elution and reversing crosslinking- Day 2 continued

1. Resuspend beads in 100ul of elution buffer.

2. Elute by placing in water bath at 65°C for 15 min. During elution, resuspend beads every 2 min by mixing briefly on a vortex mixer.

3. Spin down the beads at 16,000xg for 1 min at room temp. 

4. Place tubes on magnet, transfer 100ul supernatant to new PCR tube.

5. Wash beads with 100ul of elution buffer, and add this supernatant to the previous 100ul in PCR tube.

6. Reverse the cross-links by incubating at 65°C overnight in thermal cycler.

7. For Input samples: Thaw 100ul diluted chromatin (RIPA + lysis buffer) reserved after sonication. Add 100ul of elution buffer and mix + 10ul 10% SDS (final = 1%). Reverse the cross-links by incubating in the thermal cycler at 65°C overnight.


E. Digestion of cellular protein and RNA- Day 3

1. Prepare a mix of 200ul TE (pH 8.0) + 8ul RNAse A 10mg/ml (0.2mg/ml final); add 200ul of mix to each tube of IP and 150ul to Input samples to dilute SDS in elution buffer.

2. Mix and incubate in water bath for 2 hours at 37°C. 

3. Add 7ul of CaCl2 stock solution (300mM CaCl2 in 10mM Tris pH 8.0) to each sample, followed by 4ul of 20mg/ml proteinase K (0.2mg/ml final). 

4. Mix and incubate in a water bath at 55°C for 30 minutes.

5. Proceed with ChIP/Input DNA extraction using the Zymo research DNA clean and concentrator kit.