ChIP protocol

Preparation of mycelium

Culture

• Inoculate 6 Roux bottles of 100 ml of medium with a fresh mycelium (grown 1 or 2 days on M2 medium at 27°C).
• Incubate 62 hours in the dark at 27°C.

Fixation

• Filter the mycelium on gauze, rinse once with PBS at room temperature.
• Fix in 100ml of PBS 1X + 2.5 ml Formaldehyde 37%, shake 15 min at 27°C. Careful: Formaldehyde is toxic, work with appropriate safety equipements (gloves, chemical hood)
• Stop with 2.5 ml of Glycine 2.5 M (shake for 5 min).

Grinding and storage (In cold room)

• Filter the mycelium (the flow-through needs to be discarded in appropriate liquid trash bin)
• Wash twice with cold PBS
• Dry on wattman paper (do not hesitate to press the mycelium)
• Grind with liquid nitrogen with mortar and pestle. Careful: Use appropriate safety equipments
• Weight and store at -80°C in 50 mL Falcon tubes (transport in liquid nitrogen).
• In order to prepare ready-to-use aliquots : Weigh approximately 200 mg of mycelium (cool the spatula in liquid nitrogen before each sample, no more than 2 samples after removal of the mycelium from liquid nitrogen)

Day 1

Lysis and chromatin digestion

• Label the tubes with your name / pair number / IP or other instruction the teachers will give.
• In the ice: Resuspend the mycelium with 1 mL of Lysis buffer (+proteinase inhibitor + CaCl2)
• Mix well at least 10 times going back and forth with the P1000 and vortex. Make sure to resuspend well the mycelium. Then leave 10 min on ice, vortex again.
• Add 5μl of Micrococcale Nuclease and incubate 35 min at 37°C (while continuously shaking if possible, otherwise in a waterbath) (Include a control without MNase to check digestion). If using a waterbath, gently mix the tubes every 2 min to resuspend the mycelium.
• Stop the reaction with 30 μl of EGTA pH 8 0.5 M , incubate 5 min on ice.
• Centrifuge 3-5 min at 4°C at top speed,
• Recover the supernatant (= soluble chromatin),
• Repeat the centrifugation to remove as much as possible the unsolubilized genomic material.
• Quantify the 2µL of your chromatin (with QuBit, following the QuBit kit protocol). You may need to dilute an aliquot of the chromatin beforehand.

(Possibility of storing -80 ° C at this stage, but for the ChIP it is better to avoid the thawing steps).

Checking of chromatin fragmentation (nucleosomal digestion pattern)

• Save a 150µL aliquot of Micrococcale digested chromatin in a fresh tube, then add:
  • RNAse A (20mg/ml) 10 μl, 60 min at 37°C
  • Proteinase K (20mg/ml) 10 μl, 120 min at 65°C
  • SDS to 0.5%
  • Incubate at 65°C over night to reverse the crosslink

Day 2

Phenol extraction of the DNA

To the tube kept over-night at 65°C,

• Adjust the volume to 300 µl with H2O
• Add 300μl phenol-chloroform (Carefull! Phenol and chloroform must be used under the fume hood!)
• mix or vortex
• centrifuge 3 min (top speed, room temperature)
• recover the aqueous phase in a fresh tube
• Add 300 μl chloroform to the aqueous phase
• mix or vortex
• centrifuge 3 min (top speed, room temperature)
• recover the aqueous phase in a fresh tube

Ethanol precipitation:

• Add 1/100th of glycogen
• Add 1/10th Sodium Acetate 3 M
• Add 2.5 volume of Ethanol 100% and mix well
• Centrifuge 15 min 16000g, 4°C, carefully remove the supernatant without aspirating the pelet.
• Add 500 μl cold ethanol 70%
• Centrifuge 5 min 16000g, 4°C, carefully remove the supernatant without aspirating the pelet.
• Air dry for 10-20 min
• Resuspend in 20 μl H2O
• Add 4μl of loading buffer
• Load everything on a 1.5% agarose gel. Run 25-30 min at 100V.

It should looks like this:

ChIP:

Wash 50 µL of magnetic beads per IP, as instructed here:

• 30 µl will be used immediatly for the pre-clearing
• 20 µl will be used the next day
• the washed beads must be stored at 4°C

Dilution the chromatin :

• If needed, thaw the chromatin on ice.
• Seed 5 µg of chromatin in a DNA LoBind tube. Adjust the volume to 1.1 ml with cold Lysis buffer + protease inhibitor. Keep the tube on ice.

Prepare as many tubes as necessary for the different IPs and controls you will need

In the cold room:

• Pre-clearing: Add 30 μl of washed magnetic beads to the chromatin. Incubate between 1 hour and 4 hours at 4°C on a rotating wheel.
• Place the tubes on a magnetic rack for 2-3 min and recover the supernatant in a clean DNA LoBind tube. Keep the tube on ice.
• Save 100 μl (= Input) to a new tube and freeze it at -20°C or -80°C.
• Add the appropriate amount of antibody (see here) to the rest (1 ml). Incubate over night at 4°C on a rotating wheel.

Caution: 1 Tube (5μg) = 1 Precipitation with 1 antibody. Prepare 1 tube for each replicate of each antibody.

The tubes must be annotated this way:

Day 3

Binding and washes

• Add 20μl of washed magnetic beads to each IP and incubate 4h at 4°C on a rotating wheel.

• Washes: Place the eppendorf tube on the magnetic rack for 3 min, remove supernatant, add 1 ml of the following cold buffers and incubate 10 min on rotating wheel each time at 4°C.

  1. Lysis buffer without protease inhibitor without CaCl2 - x2
  2. Lysis buffer NaCl
  3. LiCl Washbuffer - x2
  4. Tris-EDTA

Elution

• Resuspend the beads in 62.5μL of TES preheated at 65°C and incubate at 65°C for 10 min (vortex every 2 min or shake with the thermomixer).
• Place the tube on magnet and SAVE the the supernatant in a DNA LoBind Tube.
• Repeat the elution with again 62.5 µl of warm TES
• Pool the two elutions from the same IP into the same tube.

Cross-link reversal

• Adjust the volume of Input and IP to 500 µL with TES.
• Incubate the IPs and the Input at 65°C over-night to reverse the cross-link

Day 4

DNA Purification

Use DNA LoBind tubes for every steps

• Treat the Input and IPs with:

  • RNAse A (20mg/ml): 2 μl for the IPs, 5 µl for the Inputs, 120 min at 50°C
  • Proteinase K (20mg/ml): 10 μl, 120 min at 50°C

• Phenol extraction: to the Input and IPs

  • Add: 500μl phenol-chloroform
  • mix or vortex
  • centrifuge 3 min (top speed at room temperature)
  • recover the aqueous phase in a fresh DNA LoBind tube
  • Add: 500 μl chloroform
  • mix or vortex
  • centrifuge 3 min (top speed at room temperature)
  • recover the aqueous phase in a fresh DNA LoBind tube

DNA Precipitation

• To the IPs and Input:

  • Add 1/100th of glycogen
  • Add 1/10th of Sodium Acetate 3M
  • Vortex
  • Divide into two DNA LoBind tubes (approx 250µL / tube)
  • Add 2.5 volumes Ethanol 100% (i.e. approx 625µL 100% EtOH).
  • Vortex
  • Centrifuge 10 min 16000g, 4°C, carefully remove supernatant without disturbing the pellet
  • Wash with 400µl of 70% ethanol. Spin again 10 min and remove supernatant carefully
  • Air dry 10-20 min
  • Resuspend each pellet in 15μl of Tris-EDTA. Combine the two pellets of the same IP together. Combine the two pellets of the Input together. The total volume is 30µL.

• Quantify 2µL of the Input and IP sample with the Qubit dsDNA HS kit (follow the QuBit protocol)

• Store at -20°C. Evaluate the IP specificity by qPCR and/or proceed with the library preparation.