Chromatin Immunoprecipitation (for E. coli)

 

Making crosslinked, sonicated cell lysates (“chromatin”)

 

1. Grow 2 ml o/n culture. Subculture 1/100 (e.g. 400 μl ŕ 40 ml). Grow to appropriate OD600 (typically 0.3-0.6).

 

2. Crosslink cells by adding formaldehyde to final concentration of 1% (~1 ml per 40 ml culture). Swirl flask to mix. Incubate for 20 min at room temperature.

 

3. Quench formaldehyde by adding glycine to final concentration of 0.5 M (typically 10 ml of 2.5 M glycine per 40 ml culture).

 

4. Spin cells at full speed in bench-top centrifuge. Resuspend in TBS (original culture volume). Spin again. Resuspend in 1 ml TBS. Spin in microcentrfuge, max speed, 1 min. Discard supernatant. Cells can be frozen at this point.

 

5. Resuspend cells in 1 ml FA lysis buffer containing 2 mg/ml lysoszyme. Incubate @ 37 °C for 30 min to lyse cells. Crosslinked lysates can be frozen at this point.

 

6. Chill on ice for >5 min. Sonicate to shear DNA: 2 x 30 s pulses, 50% output, keeping cells on ice. In between 30 s pulses leave cells on ice for >3 minutes.

 

7. Spin cell lysates in microcentrifuge, max speed, 5 min. Keep supernatant (should be ~1 ml). For cultures >20 ml, dilute crosslinked lysates to 1 ml per 20 ml original culture volume with FA lysis buffer, e.g. if original culture volume was 40 ml, dilute crosslinked cell lysates to final volume of 2 ml. Crosslinked, sonicated lysates can be frozen at this point.

 

Immunoprecipitation (with protein A beads)

 

1. Dilute crosslinked, sonicated lysate in FA lysis buffer such that there is 800 μl for every 10 ml original culture volume (assuming exponential phase growth).

 

2. Add 25 μl protein A beads (from 50% slurry in TBS) and appropriate amount of antibody. Incubate for 90 min at room temperature on rotisserie rotator.

 

3. Spin IP mix for 1 minute in microcentrifuge at 4,000 rpm. Remove supernatant by aspiration. Resuspend in 750 μl FA lysis buffer and transfer to Spin-X column filter.

 

4. Incubate for 3 min at room temperature on rotisserie rotator in Spin-X column. Spin (as before) and discard supernatant. Repeat wash step with FA lysis buffer, FA lysis buffer + 500 mM NaCl, ChIP wash buffer, and TE (10 mM Tris pH 8.0, 1 mM EDTA).

 

5. Resuspend beads in 100 μl ChIP elution buffer. Place Spin-X filter in fresh dolphin-nosed tube. Incubate for 10 min at 65 °C with occasional gentle mixing. Spin for 1 minute in microcentrifuge at 4,000 rpm. Discard filter.

 

6. Decrosslink by incubating o/n at 65 °C or 10 min at 100 °C (in normal 1.5 ml tube). Decrosslink input sample (i.e. crosslinked, sonicated cell lysate used for IP; typically 20 μl) as necessary.

 

7. Clean up DNA with Qiagen PCR purification kit. Use 2 μl of eluted DNA (typically from 200 μl) per 10 μl qPCR reaction.

 

 

Immunoprecipitation (with IgG beads – for TAP-tagged proteins)

 

1. Dilute crosslinked, sonicated lysate in FA lysis buffer such that there is 800 μl for every 10 ml original culture volume (assuming exponential phase growth).

 

2. Add 25 μl IgG beads (from 50% slurry in TBS). Incubate for 90 min at room temperature on rotisserie rotator. Continue from step 3 above.

 

 

Buffers

 

FA lysis buffer (150/500 mM NaCl)

50 mM Hepes-KOH, pH 7.

150/500 mM NaCl (use low salt buffer for all steps except for one wash step)

1 mM EDTA

1% Triton X-100

0.1% sodium deoxycholate

0.1% SDS

 

ChIP wash buffer

10 mM Tris-HCl, pH 8.0

250 mM LiCl

1 mM EDTA

0.5% Nonidet-P40

0.5% sodium deoxycholate

 

ChIP elution buffer

50 mM Tris-HCl, pH 7.5

10 mM EDTA

1% SDS