TITLE: Immunoprecipitation, Decross-linking, and DNA Extraction
AUTHORS: Vasso Makrantoni, Daniel Robertson, Adele L. Marston

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.</div></div>

[STEPS]
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Prewash (n × 15) μl of Protein G Dynabeads (n = number of IP samples) in  containing 0.1% SDS,  and protease inhibitors with rotation for . Place the microcentrifuge tubes on a magnet and discard the supernatant. (1/3)
[ice-cold 1× FA buffer]
[PMSF]
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Prewash (n × 15) μl of Protein G Dynabeads (n = number of IP samples) in  containing 0.1% SDS,  and protease inhibitors with rotation for . Place the microcentrifuge tubes on a magnet and discard the supernatant. (2/3)
[ice-cold 1× FA buffer]
[PMSF]
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Prewash (n × 15) μl of Protein G Dynabeads (n = number of IP samples) in  containing 0.1% SDS,  and protease inhibitors with rotation for . Place the microcentrifuge tubes on a magnet and discard the supernatant. (3/3)
[ice-cold 1× FA buffer]
[PMSF]
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Add the antibody against the protein of interest to  (step 13 in protocol "Cell Lysis and Sonication") and  and mix.
[chromatin extract]
[prewashed Dynabeads]
The amount of SDS in immunoprecipitation can interfere with antibody binding efficiency therefore, lower amount of SDS can also be used. Either omit SDS in 2xFA buffer or reduce SDS added afterward to a final concentration of 0.05% before chromatin immunoprecipitation.
The amount of antibody added should be in excess of the protein being immunoprecipitated and should be determined empirically. We use the following amounts of antibodies per IP:    (Tonbo Biosciences)  (Roche)  (Bio-Rad)   (Roche)   (Sigma)No blocking and preclearing is required for the magnetic beads.
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Incubate on a rotating wheel at .
4 °C
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Place the tubes on the magnet and discard the supernatant. Perform the following washes, using 1 ml per sample of Wash buffer on a rotating wheel for 5 min at room temperature. Discard supernatants after each wash.
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Wash using  on a rotating wheel for  at . Discard supernatant after wash.
[ChIP Wash buffer 1 - low salt]
4 Room temperature
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Wash using  on a rotating wheel for  at . Discard supernatant after wash.
[ChIP Wash buffer 2 - high salt]
4 Room temperature
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Wash using  on a rotating wheel for  at . Discard supernatant after wash.
[ChIP Wash buffer 3]
4 Room temperature
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Wash using  on a rotating wheel for  at . Discard supernatant after wash.
[ChIP Wash buffer 4]
4 Room temperature
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Following the final wash, place the samples on magnetic rack and discard the supernatant without disturbing the beads.
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Move forward with: reversal of cross-linking and isolation of DNA for qPCR, ORChIP-seq preparation after the final wash
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Add  in sterile water Chelex-100 resin directly to the washed Dynabeads (IP sample) and to  (see step 14 from protocol "Cell Lysis and Sonication"). Keep the Chelex beads in suspension while pipetting.
[10% slurry (wt/vol)]
[thawed “Input” samples]
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Briefly vortex samples to mix the slurry and boil for .
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Cool the tubes to  and quickly centrifuge condensate to the bottom of the tube at . Proceed immediately to the next step.
0 Room temperature
Centrifuge: 400 34
Variation in the amount of beads added can affect the specific signal–background ratio. Make sure to keep the slurry suspended while distributing.
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Add  to each sample and incubate on a heat block at  for  with occasional mixing to resuspend beads.
[Proteinase K (10 mg/ml)]
55 °C
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Boil the samples at  for  to inactivate the Proteinase K.
100 °C
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Spin the tubes briefly at  and carefully transfer approximately , termed the IP sample in a new tube.
Centrifuge: 400 34
[DNA supernatant]
Make sure not to transfer any Chelex resin as it can lead to loss of PCR signal.
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Store samples at . (PAUSE POINT).
-20 °C
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Purified DNA from step 12 can be used in qPCR, following the subsequent steps. We use Luna Universal Probe qPCR Master Mix in a 10 μl reaction in a 96-well plate and run the following program:
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Set up the following reaction mix:  (for Input use 1:300, for IP use 1:6 dilutions) (20 μM each)ddH2O up to 10 μl final volume
[DNA Template]
[primer pair]
[master mix]
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Run the following program:   ABCD1StepTemperatureTimeCycles2Initial denaturation95 °C1 min3Denaturation95 °C15 sec42 cycles4Extension60 °C30 sec5Melt curveaccording to Lightcycler 480 recommendationsaccording to Lightcycler 480 recommendations. according to Lightcycler 480 recommendations.
ABCD1StepTemperatureTimeCycles2Initial denaturation95 °C1 min3Denaturation95 °C15 sec42 cycles4Extension60 °C30 sec5Melt curveaccording to Lightcycler 480 recommendationsaccording to Lightcycler 480 recommendations. according to Lightcycler 480 recommendations.
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Perform data analysis using Microsoft Excel software using the following formulas:Determine the ChIP enrichment (i.e., ChIP/Input value), by calculating ΔCt using the following formula: ΔCt = Ct^{ChIP} − (Ct^{Input} − log_{primer efficiency} (Input dilution factor))From this, the ChIP enrichment is calculated using: ChIP/Input = (primer efficiency)^{(−ΔCt)}    where Ct values are mean threshold cycles of PCR performed in triplicate per DNA sample.
To increase accuracy and reproducibility, we advise the use of an electronic dispensing pipette for qPCR. For primer design we recommend the use of Prime3Plus software. In the general settings of the software, we use product size range 70–200 bp (optimal amplicon size ~120 bp), optimal primer size 20 bp in length and optimal primer Tm for use with Luna Universal Probe qPCR Master Mix should be designed to anneal at 60 °C, with optimal primer GC% between 50% and 60%. Once primers are designed determine their efficiency and specificity using genomic DNA prior to performing qPCR with ChIP DNA sample. Finally, also include a No Template Control (NTC) in the reaction, where no amplification and no melting curve should be generated.
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
To determine whether the amount of immunoprecipitated DNA is sufficient for library preparation for ChIP-seq, quantify  using the Qubit HS kit.
[ChIP products and Input]
Most commonly, immunoprecipitated DNA yields are in the range of 2–10 ng.
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Pool magnetic beads of the same IP samples together and resuspend in .
[TES buffer]
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Elute the immunoprecipitated chromatin by incubating the resuspended beads in TES buffer at  for
65 °C
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Place tubes on magnetic rack and transfer the supernatant, termed the IP sample, to a new LoBind DNA tube.
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Add  to  in a LoBind DNA tube.
[TES]
[pooled Input samples]
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Decross-link both IP and Input samples at .
65 °C
[(minimum 6 hours)]
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Cool the samples and add  for  at  to degrade RNA.
[RNAse (10 mg/ml)]
37 °C
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Add  per  to both IP and Input samples and incubate at for .
[10 mg/ml Proteinase K]
[solution]
65 °C
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
Purify ChIP-seq library by using Wizard SV purification kit according to manufacturer’s instructions. Proceed to the next step.
?. [Immunoprecipitation, Decross-linking, and DNA Extraction]
To determine whether the amount of immunoprecipitated DNA is sufficient for library preparation for ChIP-seq, quantify  using the Qubit HS kit.
[ChIP products and Input]
Most commonly, immunoprecipitated DNA yields are in the range of 2–10 ng.