TITLE: Chromatin Immunoprecipitation (ChIP) Assay Protocol
AUTHORS: Sam Li

[DESCRIPTION]
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[STEPS]
?. [Chromatin Sample Preparation]
Culture between 1-15 million cells. Collect cells by spinning down at 500xg at 4°C for 5 minutes. Wash cells with PBS at room temperature.
?. [Chromatin Sample Preparation]
Remove the PBS and add freshly made basic cell culture media (it should not contain any serum or large molecular weight proteins to avoid any interferences from those proteins) containing 1% formaldehyde (CH2O) to cross-link the DNA-protein complexes.
?. [Chromatin Sample Preparation]
Incubate for 10 minutes at room temperature with gentle agitation on a rocking platform.
?. [Chromatin Sample Preparation]
Remove the fixation solution by spinning down at 500xg at 4°C for 5 minutes. To quench the reaction, for cell count of 1-5 million, add 3mL of 0.65M glycine solution; for cell count of 5-10 million, add 4mL of 0.65M glycine solution; for cell count of 10-15 million, add 5mL of 0.65M glycine solution.
?. [Chromatin Sample Preparation]
Incubate at room temperature for 5 minutes with gentle agitation on a rocking platform.
?. [Chromatin Sample Preparation]
Remove the glycine solution by spinning down at 500xg at 4°C for 5 minutes. Discard the supernatant. The pellet can be frozen at -80°C after adding 1µL of Protease Inhibitor Cocktail (PIC).
?. [Chromatin Sample Preparation]
To lyse the cells, for cell count of 1-5 million, add 0.4mL of Hypotonic Buffer; for cell count of 5-10 million, add 0.8mL of Hypotonic Buffer; for cell count of 10-15 million, add 1mL of Hypotonic Buffer to the cell pellet. Resuspend the cells and incubate at 4°C for 10 minutes.
?. [Chromatin Sample Preparation]
Centrifuge the hypotonic slurries at 5,000xg at 4°C for 5 minutes to collect the nuclei.
?. [Chromatin Sample Preparation]
For cell count of 1-5 million, add 0.3mL of Digestion Buffer; for cell count of 5-10 million, add 0.4mL of Digestion Buffer; for cell count of 10-15 million, add 0.5mL of Digestion Buffer to the nuclei, immediately followed by adding 2µL of PIC to each sample.
?. [Chromatin Sample Preparation]
Add Micrococcal Nuclease to each sample to digest the DNA. Mix by inverting the tube several times and incubate at 37°C for 20 minutes. Mix by inversion every 3-5 minutes. The amount and incubation time of Micrococcal Nuclease required to digest the genomic DNA to an optimal 150 900 bp length may need to be determined empirically for individual cell types.
?. [Chromatin Sample Preparation]
Stop digestion by adding 10µL of 0.5M EDTA per sample and place the sample on ice.
?. [Chromatin Sample Preparation]
Pellet nuclei by centrifugation at 12,000xg at 4°C for 1 minute. Discard the supernatant.
?. [Chromatin Sample Preparation]
Resuspend nuclear pellet in Lysis Buffer and incubate the sample on ice for 10 minutes to lyse the nuclei. Alternatively, sonicate to shear the DNA. The time and strength for sonication may need to be determined empirically.
?. [Chromatin Sample Preparation]
Centrifuge the sample at 16,000xg at 4°C for 10 minutes. Transfer the supernatant to a clean dry microcentrifuge tube.
?. [Chromatin Sample Preparation]
Add 1µL of PIC to each sample and mix.
?. [Chromatin Sample Preparation]
Chromatin sample is now ready for ChIP Assay. If the sample is not to be used immediately, store at -80°C. It is recommended that the shearing efficiency is analyzed at this stage to ensure that 150-900bp fragments are obtained during shearing the DNA to increase the ChIP efficiency.
?. [Addendum: Shearing Efficiency Analysis]
i. Take 50µL aliquot of sheared chromatin from each sample into a new microcentrifuge. Add 5µL of 1M NaHCO3, 5µL of 5M NaCl and 50µL of Distilled Water. Mix thoroughly and incubate at 65°C (heat block) for two hours or incubation can be extended for overnight.
?. [Addendum: Shearing Efficiency Analysis]
ii. Remove the sample from heat block, add 1µL of Proteinase K to each sample, vortex briefly and perform a spin down at 500xg at 4°C for 5 minutes. Incubate at 37°C for one hour.
?. [Addendum: Shearing Efficiency Analysis]
iii. Add 2µL of Proteinase K Stop Solution to each sample, vortex briefly and perform a short spin down.
?. [Addendum: Shearing Efficiency Analysis]
iv. Purify the DNA by QIAquick™ PCR purification kit, according to manufacturer’s manual.
?. [Addendum: Shearing Efficiency Analysis]
v. Quantify the DNA in the sample using a Nanodrop. This will be used to determine the volume of chromatin to load in ChIP assay.
?. [Addendum: Shearing Efficiency Analysis]
vi. To ensure 150-900bp fragments have been obtained during shearing the DNA, run each DNA sample on a 1.3-1.5% agarose gel and visualize against a marker of known size DNA fragments (e.g., 100bp ladder).
?. [Chromatin Immunoprecipitation Assay]
Mix the Chromatin Sample, Protease Inhibitor Cocktail (PIC), optimal quantity of BioLegend’s Go-ChIP-GradeTM Purified antibodies and add Column Conditioning Buffer to make final 1mL slurries. Gently rotate at 4°C for one hour or incubation can also be extended to overnight.
?. [Chromatin Immunoprecipitation Assay]
Prepare the high-throughput (HT) Protein A or G 96 well plate or Spin Column by adding 600µL of Column Conditioning Buffer in each well or column and allow it to flow through via gravity (~15 minutes).
?. [Chromatin Immunoprecipitation Assay]
Discard the flow-through and repeat steps 18 and 19.
?. [Chromatin Immunoprecipitation Assay]
Remove the slurries from the rotator following 4°C incubation and briefly spin down to remove residual liquid from the caps.
?. [Chromatin Immunoprecipitation Assay]
Load the entire 1mL slurries and allow to flow completely through the high-throughput (HT) Protein A or G 96 well plate or Spin Column at room temperature (approximate 15-20 minutes).
?. [Chromatin Immunoprecipitation Assay]
Add 600µL of Wash Buffer 1 to each well or column and centrifuge at 2,000xg for plate, or 4,000xg for column, for one minute at room temperature. Discard the flow through and repeat once.
?. [Chromatin Immunoprecipitation Assay]
Add 600µL of Wash Buffer 2 to each well or column and centrifuge at 2,000xg for plate, or 4,000xg for column, for one minute at room temperature. Discard the flow through and repeat once.
?. [Chromatin Immunoprecipitation Assay]
Add 600µL of Wash Buffer 3 to each well or column and centrifuge at 2,000xg for plate, or 4,000xg for column, for one minute at room temperature. Discard the flow through and repeat once.
?. [Chromatin Immunoprecipitation Assay]
Spin dry at 4,000xg for the plate or 16,000xg for the column for one minute at room temperature to remove any remaining liquid from the membrane of the plate or column. Place a clean 96 well collection plate or a collection tube beneath the plate or column.
?. [Chromatin Immunoprecipitation Assay]
Add 50µL of Elution Buffer to each well or column. Incubate at room temperature for 15 minutes.
?. [Chromatin Immunoprecipitation Assay]
Centrifuge the plate or column at 4,000xg for the plate, or 16,000xg for the column, for one minute at room temperature to collect the eluted chromatin-protein complex.
?. [Chromatin Immunoprecipitation Assay]
To each eluted sample, add 5µL of 1M NaHCO3, 5µL of 5M NaCl and 50µL of Distilled Water. Mix thoroughly and incubate at 65°C on the heat block for two hours or the incubation time can be extended to overnight.
?. [Chromatin Immunoprecipitation Assay]
Remove the sample from the heat block, add 1µL of Proteinase K to each sample, vortex briefly and perform a short spin down. Incubate at 37°C for one hour.
?. [Chromatin Immunoprecipitation Assay]
Add 2µL of Proteinase K Stop Solution to each sample, vortex briefly and perform a short spin down.
?. [Chromatin Immunoprecipitation Assay]
Purify the DNA by QIAquick™ PCR purification kit, according to manufacturer’s manual. Purified DNA is ready for downstream real-time qPCR analysis.