Sep 19, 2023
Double-fixation prior to ChIPs
- 1Oklahoma Medical Research Foundation;
- 2University of Oklahoma Health Sciences Center
Protocol Citation: Jacob G Kirkland, Mary Bergwell, JinYoung Park 2023. Double-fixation prior to ChIPs. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm396nl3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 19, 2023
Last Modified: September 19, 2023
Protocol Integer ID: 88036
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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.
Materials
Disuccinimidyl glutarate (DSG) [ChemCruz]
DMSO [Sigma]
PBS [Fisher Scientific]
2.5M Glycine Solution: Dissolve 93.84g Glycine (MW: 75.07g/mol) in 400ml sterile water. Adjust final volume to 500 ml and sterile filter
CiA Fix Buffer: 25 ml 1M HEPES pH 8.0; 1 ml 0.5M EDTA; 0.5 ml 0.5M EGTA; 10 ml 5M NaCl Adjust final volume to 500 ml with sterile water and filter sterilize
trypsin-EDTA 0.25% (Fisher Scientific)
16% Formaldehyde Solution (w/v) Methanol-Free [Thermo Scientific]
Safety warnings
Use proper safety precautions when using formaldehyde.
Fresh DSG
Fresh DSG
30m
30m
Place DSG at room temperature for approximately 30 minutes before weighing it out.
30m
Measure DSG and dissolve in DMSO accordingly (using approximately 80 ul of solution per sample)
| Number of Samples | DSG (mg) | Vol. DMSO (ul) | |
| 2 | 13 | 160 | |
| 4 | 25 | 320 | |
| 6 | 38 | 480 | |
| 12 | 72 | 960 |
5m
Isolate and count single cells
Isolate and count single cells
20m
20m
Harvest the cells by first washing with PBS
1m
Incubate cells in trypsin-EDTA 0.25% until dissociated
5m
Quench trypsin with media + FBS and move cells to a 15ml conical. Spin down at 300g for 4 minutes
4m
Resuspend cells in PBS and count
5m
Move 30e6 cells to a new tube and bring the volume up to 10mls with PBS and spin down at 300g for 4 minutes
4m
Resuspend cells in 10mls fresh PBS
1m
DSG fixation (First)
DSG fixation (First)
36m
36m
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
30m
Pellet samples by spinning at 1500 x g for 10 minutes at 4C
4m
Carefully aspirate the supernatant, taking care not to disturb the pellet.
1m
Resuspend each sample in 10 ml of CiA Fix Buffer
1m
Formaldehyde Fixation (Second)
Formaldehyde Fixation (Second)
34m
34m
Add 667 ul of 16% Formaldehyde to the sample dropwise and incubate for exactly 10 minutes at room temperature using rotation
10m
Stop cross-linking by adding 555 ul of 2.5M glycine.
1m
Incubate samples on ice for 5 minutes
5m
Pellet samples by spinning at 1500 x g for 10 minutes at 4°C
10m
Aspirate supernatant taking care not to disturb the pellet
1m
Wash fixed cells with 10ml PBS
1m
Pellet samples by spinning at 1000 x g for 5 minutes at 4C and aspirate supernatant.
5m
Samples can be stored at -80°C (snap freeze) or can begin processing for chromatin immunoprecipitation
1m
Protocol references
Adapted from B.Tian et al., 2012, pp. 106-108
DOI: 10.1007/978-1-61779-376-9_7