Dec 26, 2025
Chromatin immunoprecipitation (ChIP) in 22Rv1 cells
- Xin Xu1,
- Yujuan Wang1
- 1Princess Margaret Cancer Center
Protocol Citation: Xin Xu, Yujuan Wang 2025. Chromatin immunoprecipitation (ChIP) in 22Rv1 cells. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly5dzevx9/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: December 22, 2025
Last Modified: December 26, 2025
Protocol Integer ID: 235595
Keywords: chromatin immunoprecipitation, ChIP, ChIP-seq, epigenetics, transcription regulation, histone modification, protein-DNA interaction, cancer cells, chromatin biology, dna purification, cells this protocol, chip, cell, chd9 knockdown, purification
Disclaimer
This protocol is provided for research use only and may require optimization depending on experimental conditions.
Abstract
This protocol describes chromatin immunoprecipitation (ChIP) in 22Rv1 cells with or without CHD9 knockdown, followed by DNA purification and ChIP-seq library preparation.
Materials
Cells
- 22Rv1 cells (control and CHD9 knockdown)
- 5 x 10^6 cells per ChIP reaction
Reagents
- Formaldehyde (to 1% final concentration)
- Glycine (to 125 mM final concentration)
- PBS (cold)
Modified RIPA buffer (pH 8)
- 10 mM Tris-HCl, pH 8
- 1 mM EDTA
- 140 mM NaCl
- 1% Triton X-100
- 0.1% SDS
- 0.1% sodium deoxycholate
- Protease inhibitor (add fresh)
High-salt modified RIPA buffer
- Same as modified RIPA buffer, with NaCl adjusted to 500 mM
LiCl wash buffer (pH 8)
- 10 mM Tris-HCl, pH 8
- 1 mM EDTA
- 250 mM LiCl
- 0.5% NP-40
- 0.1% sodium deoxycholate
TE buffer
- 10 mM Tris-HCl, pH 8
- 1 mM EDTA
Decrosslinking buffer
- 1% SDS
- 0.1 M NaHCO3
Antibodies
- Anti-CHD9 (Proteintech, 13402-1-AP)
- Anti-H3K27ac (Abcam, ab4729)
Beads
- Protein A magnetic beads (Thermo Fisher, 10002D)
- Protein G magnetic beads (Thermo Fisher, 10004D)
Enzymes and kits
- RNase A (Thermo Fisher, EN0531)
- Proteinase K (Thermo Fisher, AM2546)
- PCR purification kit (QIAGEN, 28004)
- ThruPLEX DNA-seq kit (Takara, R400676)
Equipment
- Bioruptor sonicator (Diagenode)
- End-over-end rotator (4°C)
- Thermomixer or shaking incubator
- Magnetic rack or centrifuge
- Microcentrifuge
Troubleshooting
Before start
- All buffers should be freshly prepared or thawed on ice.
- Add protease inhibitor to lysis buffers immediately before use.
- Perform all steps on ice or at 4°C unless otherwise indicated.
- Pre-chill centrifuges, rotators, and sonicator to 4°C.
- Use low-retention, DNA-free tubes and tips.
Crosslinking
20m
Harvest 5 x 10^6 22Rv1 cells per sample.
Add formaldehyde to a final concentration of 1%, and incubate for 10 min at room temperature.
10m
Quench crosslinking by adding glycine to a final concentration of 125 mM.
5m
Pellet cells and wash twice with cold PBS, and then discard supernatant completely.
5m
Cell lysis and chromatin shearing and pre-cleaning
1h 30m
Resuspend cell pellet in 300 µL modified RIPA buffer supplemented with protease inhibitor.
Sonicate chromatin using a Bioruptor (Diagenode) at 4°C:
18 cycles
30 s ON / 30 s OFF
30m
NOTE: Sonication conditions may require optimization depending on cell density and instrument performance.
Briefly centrifuge to remove insoluble debris and transfer supernatant to a new tube.
Add 40 µL protein A/G beads to the sonicated chromatin. Incubate with rotation at 4°C. Transfer the pre-cleared chromatin to a new tube.
1h
Immunoprecipitation
18h 30m
For each ChIP, prepare antibody-coated beads:
- 5 µg antibody
- 11 µL Protein A beads
- 11 µL Protein G beads
Incubate with rotation at 4°C for at least 6 h.
6h
Combine pre-cleared chromatin with antibody-coated beads. Incubate overnight at 4°C with rotation.
12h
Wash beads at 4°C, rotating for 5 min per wash:
- Once with 0.5 mL modified RIPA buffer
- Once with 0.5 mL high-salt modified RIPA buffer (500 mM NaCl)
- Once with 0.5 mL LiCl buffer
- Twice with 0.5 mL TE buffer (pH 8)
Between washes, collect beads and carefully remove supernatant.
30m
Elution and decrosslinking
13h
Resuspend beads in 100 µL decrosslinking buffer. Add 1 µL RNase A. Incubate at 37°C for 30 min with shaking.
30m
Add 2 µL Proteinase K. Incubate at 55°C for 30 min with shaking.
30m
Reverse crosslinks by incubating at 65°C overnight with shaking.
12h
DNA purification and library preparation
2h 30m
Purify DNA using the QIAGEN PCR purification kit (28004) according to the manufacturer’s instructions. Elute purified ChIP DNA in nuclease-free water or elution buffer.
30m
Use purified DNA for Illumina ChIP-seq library construction with the ThruPLEX DNA-seq kit (Takara, R400676) following the manufacturer’s protocol.
2h