TITLE: Plant Chromatin Immunoprecipitation
AUTHORS: Laura Poza-Viejo, Ivan del Olmo, Pedro Crevillén

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>Chromatin Immunoprecipitation (ChIP) is a crucial technique to study chromatin regulation, epigenetic phenomena and transcription factor DNA-binding in vivo. This technique is widely used in model plant systems like Arabidopsis but reliability in other plant systems is challenging. We adapted our well-established Arabidopsis ChIP protocol to be used with leaves and inflorescences of Brassica crops species (</span><span style = "font-style:italic;">Brassica rapa</span><span> and </span><span style = "font-style:italic;">Brassica napus</span><span>). This protocol is  based on Gendrel et al. </span><span style = "font-style:italic;">Nature Methods </span><span>volume 2, pages 213–218 (2005), but has several modifications including chromatin sonication using a BioRuptor, immunoprecipitation using magnetic Dynabeads, and DNA elution is performed using Chelex 100 for ChIP-PCR or magnetic SPRI beads for ChIP-seq. The protocol is quick and very reproducible and has been validated against a number of histone modifications and protein tag antibodies. In this new version V2 we describe how to purify and recover DNA after immunoprecipitation if DNA is used for next-generation sequencing library preparation in ChIP-seq experiments. </span></div><style>
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[STEPS]
?. [Harvest and crosslinking]
Harvest 1-2 g of Arabidopsis or Brassica seedlings/leaf tissue/inflorescence in a 50ml conical centrifuge tube.
?. [Harvest and crosslinking]
Add Phosphate-buffered saline buffer (PBS) with 1% formaldehyde (FAA)1.To detect histone modifications this is enough, but for protein binding we perform a dual crosslinking with DSG (disuccinimidyl glutarate) 2 mM.
?. [Harvest and crosslinking]
Stop the crosslinking by adding Glycine 2 M to a final concentration of 0.125 M. (2.5 ml of 2 M Glycine in 37 ml of water), vacuum for 5 min.
?. [Harvest and crosslinking]
Remove the tissue from the vacuum and rinse off the formaldehyde with MilliQ water twice. Remove as much water as possible from the seedlings by placing on a paper towel.
?. [Harvest and crosslinking]
Freeze in liquid nitrogen and store crosslinked tissue at -80°C
?. [Chromatin extraction]
Grind with mortar in liquid nitrogen to a fine powder.
?. [Chromatin extraction]
Pass to a 50 ml centrifuge tube and add the powder to 10 ml of Extraction Buffer 1. After adding buffer, ensure you mix well so all the tissue has dissolved into the buffer.
?. [Chromatin extraction]
Filter the solution through a layer of Miracloth into a 50ml centrifuge tube. Repeat step 9 until the solution is clear of big tissue particles.
?. [Chromatin extraction]
Centrifuge at 5000 xg for 10 min at 4°C.
?. [Chromatin extraction]
Gently remove supernatant and resuspend the pellet in 1 ml of Extraction Buffer 2. Transfer the solution to 1.5 ml centrifuge tube.
?. [Chromatin extraction]
Centrifuge at 5000 xg for 10 min at 4°C.
?. [Chromatin extraction]
Remove supernatant and resuspend pellet in 300 µl of Extraction Buffer 3.
?. [Chromatin extraction]
In a clean eppendorf, add 600 µl of Extraction Buffer 3. Take the 300 µl solution (resuspended pellet) from step 13 and carefully lay it on top of the clean 600 µl of Extraction Buffer 3.
?. [Chromatin extraction]
Centrifuge at 16000 xg for 60min at 4 °C. At this point, it is advisable to prepare Nuclei Lysis Buffer and ChIP Dilution Buffer, to turn on the Bioruptor and to prepare the Dynabeads (go to step 20).
?. [Chromatin extraction]
Remove the supernatant and resuspend the chromatin pellet in 200 µl of Nuclei Lysis Buffer3.
?. [Chromatin extraction]
Resuspend the pellet by pipetting up and down (keep solution cold). Take a 5 µl aliquot of each sample to check sonication efficiency4.
?. [Immunoprecipitation]
Prepare the Dynabeads magnetic beads (Protein A for rabbit antibody or Protein G for mouse antibody). For each immunoprecipitation wash 15 µl Dynabeads in 500 µl ChIP dilution buffer twice. Then, add 1-5 µg of antibody and incubate the Dynabeads and the antibody in 50 µl ChIP Dilution Buffer rotating at 4°C for 1 h.
?. [Immunoprecipitation]
Wash prepared antibody coated Dynabeads with 500 µl of ChIP dilution buffer. Let Dynabeads attach to the magnet. Then, discard buffer. Resuspend Dynabeads in 50µl of ChIP Dilution Buffer and keep aside on ice.
?. [Immunoprecipitation]
For the immunoprecipitation, measure chromatin solution from step 19, usually ~150 µl. Take INPUT: 10% of the volume (15 µl) of chromatin and store it at -20°C.
?. [Immunoprecipitation]
Dilute chromatin solution 10 times adding 1.5 ml of ChIP Dilution Buffer. The point here is to dilute the 1% SDS to 0.1% SDS with ChIP dilution buffer. This is enough for two immunoprecipitations or one immunoprecipitation and no-antibody control sample. For three immunoprecipitations is better to start from 300 µl of Nuclei Lysis Buffer (step 16).
?. [DNA recovery for ChIP-PCR]
Take the INPUTs frozen the day before and elute immune complexes and INPUTs by adding 200 µl 10% Chelex 100 resin (10 g in 100 ml water) and boil the samples at 98°C for 10 min with agitation in a thermomixer.
?. [DNA recovery for ChIP-PCR]
Cool in ice and incubate with 2 µl Proteinase K (10 mg/ml) at 37°C for 20 min.
?. [DNA recovery for ChIP-PCR]
Boil samples again at 98°C for 10 min with agitation in thermomixer to inactivate Proteinase K.
?. [DNA recovery for ChIP-PCR]
Cool in ice and spin the tubes in microcentrifuge at 16000 xg for 5 min at 4°C. Transfer ~160 µl of the supernatant to a new DNA low binding microtube. Do not take any Chelex particle. Store at 4°C or at 20-°C for long term storage.
?. [DNA recovery for ChIP-PCR]
For real-time quantitative PCR analysis, ChIP DNA can be used directly but INPUT DNA must be diluted at least 1:10 in water7. Usually 1-2 µl of the DNA solution is enough for PCR amplification.
?. [Harvest and crosslinking]
Crosslink in a desiccator attached to a vaccum pump for 5 + 5 min (for Arabidopsis seedlings and inflorescences) or 5 min + 10 min + 10 min (for Brassica leaves and inflorescences)2.
?. [Immunoprecipitation]
Add antibody coated Dynabeads into the 750 μl of chromatin solution in a DNA low binding microtube. Incubate the immunoprecipitation rotating at 4°C overnight6.
?. [Immunoprecipitation]
The next morning perform the washes using 1 ml of each buffer (see below). Wash at 4°C for 5 min with gentle rotation and let beads attach to magnets in between washes. Use the sequence of buffers listed below:a) Low Salt Wash Buffer (2 washes)b) High Salt Wash Buffer (1 wash, optional)c) LiCl Wash Buffer (1 wash, optional)d) TE Buffer (2 washes)
?. [DNA recovery for ChIP-seq]
Prepare the Elution buffer. Elute immune complexes by adding 50 μl of fresh Elution Buffer to the pelleted beads. Vortex briefly to mix and incubate at 65 °C for 15 min with gentle agitation. Let beads attach to magnet and carefully transfer 40 μl of the supernatant fraction (eluate) to another tube and repeat elution. Combine the two eluates (Final volume = 80 μl).
?. [DNA recovery for ChIP-seq]
Take the INPUTs frozen the day before.  Add 3.2 μl 5 M NaCl to each ChIP eluate and to the INPUTs and reverse crosslink incubating overnight at 65 °C.
?. [DNA recovery for ChIP-seq]
Next day, add 180 µl (2.2x vol.) of SPRI beads. Carefully mix by pipetting up and down (at least ten times) and incubate for 5 min at room temperature. Separate supernatant from beads by placing the tubes in the magnet for 5 min. Discard supernatant but leave tubes on magnet.
?. [DNA recovery for ChIP-seq]
Elute DNA from SPRI beads in 30 µl of Tris-HCl 10 mM pH 8. Mix by pipetting at least ten times and incubate for 2 min.
?. [DNA recovery for ChIP-seq]
Place tube on magnet and incubate for 5 min, and finally transfer supernatant to a new tube (aprox. 25 µl). DNA concentratin can be determined using a Qubit fluorometer. Usually 1-10 µl are used for ChIP-seq library preparation.
?. [DNA recovery for ChIP-seq]
Wash SPRI beads with 100 µl of 70% ethanol incubating or 30 sec, without disturbing the beads, then discard supernatant. Repeat wahses four times.
?. [DNA recovery for ChIP-seq]
Move tubes off the magnet and air dry the SPRI beads for 4 min at room-temperature.