Double-fixation prior to ChIPs
Jacob G Kirkland, Mary Bergwell, JinYoung Park
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Abstract
This protocol is for the double-fixation of chromatin regulators for chromatin precipitation. The double-fixation method can improve signal-to-noise ratios of chromatin regulator ChIPs, especially for transiently bound proteins or those found in large protein complexes.
Steps
Fresh DSG
Place DSG at room temperature for approximately 30 minutes before weighing it out.
Measure DSG and dissolve in DMSO accordingly (using approximately 80 ul of solution per sample)
| A | B | C |
|---|---|---|
| Number of Samples | DSG (mg) | Vol. DMSO (ul) |
| 2 | 13 | 160 |
| 4 | 25 | 320 |
| 6 | 38 | 480 |
| 12 | 72 | 960 |
Isolate and count single cells
Harvest the cells by first washing with PBS
Incubate cells in trypsin-EDTA 0.25% until dissociated
Quench trypsin with media + FBS and move cells to a 15ml conical. Spin down at 300g for 4 minutes
Resuspend cells in PBS and count
Move 30e6 cells to a new tube and bring the volume up to 10mls with PBS and spin down at 300g for 4 minutes
Resuspend cells in 10mls fresh PBS
DSG fixation (First)
Add 80 ul DSG stock to each sample in 10 ml of PBS and incubate for 30 minutes at room temperature with rocking.
Note: After incubation with DSG, cells may adhere to the walls of pipette tips, so following
this incubation, it is recommended that tips be coated with 1% BSA/PBS
Pellet samples by spinning at 1500 x g for 10 minutes at 4C
Carefully aspirate the supernatant, taking care not to disturb the pellet.
Resuspend each sample in 10 ml of CiA Fix Buffer
Formaldehyde Fixation (Second)
Add 667 ul of 16% Formaldehyde to the sample dropwise and incubate for exactly 10 minutes at room temperature using rotation
Stop cross-linking by adding 555 ul of 2.5M glycine.
Incubate samples on ice for 5 minutes
Pellet samples by spinning at 1500 x g for 10 minutes at 4°C
Aspirate supernatant taking care not to disturb the pellet
Wash fixed cells with 10ml PBS
Pellet samples by spinning at 1000 x g for 5 minutes at 4C and aspirate supernatant.
Samples can be stored at -80°C (snap freeze) or can begin processing for chromatin immunoprecipitation
