MNase-ChIP

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Note: This is a preliminary draft of this protocol, and has not yet been tested. ChIP should be preformed after MNase digestion and before Protein and RNA digestion. Use appropriate antibody for protein being immuno-precipitated, see Chromatin Immunoprecipitation From Germinating Conidia and the MNase Protocol for additional details and notes that may not have been reproduced here. Elements of the MNase Protocol w/ out Cross-linking could also be utilized in the place of the MNase Protocol if desired


Contents

Pre-Preparation

Preparation of Solid Culture Media

  1. Prepare one Flask with 50mL of Solid Culture Media for each strain to be used in the experiment

See: Solid Media Preparation for details

Preparation of Liquid Culture Media

  1. Prepare one Flask with 25mL of Liquid Culture Media for each media/strain combination to be used

See: Liquid Media Preparation for details


Inoculation of Stock Cultures

  1. 6 days before the start of the experiment, inoculate 2 flasks of 50mL Solid Culture Media with each Strain to be used in the experiment utilizing sterile technique.

See: Inoculation of Stock Cultures for details


Preparation of Reagents

  1. Prepare all stock reagents and daily use aliquots as needed (see Recipes section below).


Day 1

Preparation of Conidial Suspension

  1. Prepare a conidial suspension from each fully developed per-prepared solid media culture to be used in this experiment

See: Conidial Suspension Preparation for details


Inoculation of Liquid Cultures

See Inoculation of Liquid Cultures for details

Example: If your are using 3 strains (WT and 2 Mutant strains), inoculating one flask of Liquid Media with each strain would result in a total of 3 inoculated liquid cultures.


Harvesting Germlings

See Harvesting Neurospora Germlings for details


Cross-linking

  1. Move cell suspension to a 125mL Erlenmeyer flask and add 270uL of 37% Formaldehyde (final Formaldehyde concentration = 1%)
  2. Incubate on rotating platform at room temp for 30min
  3. Add 500uL 2.5M glycine to quench the Formaldehyde
  4. Transfer cell suspension to a new 50mL Screw Top Falcon Tube
  5. Centrifuge at 3000 RPM for 10 min to pellet cells and then pipette off and discard the supernatant.
  6. Add 40mL of 1X PBS buffer and re-suspend the cells.
  7. Centrifuge at 3000 RPM for 10 min to pellet cells and then pipette off and discard the supernatant.
  8. Add 1mL ice cold PBS and re-suspend cells.


Lysing Cells / Chromatin

  1. Aliquot each cell suspensions into two parts and transfer into two sterile 1.5mL epi-tubes.
  2. Centrifuge at 5000 RPM for 5 min at 40C and then pipette off and discard the supernatant.
  3. Add 1mL ice cold NPS buffer with Calcium Chloride (after adding PMSF and Protease Inhibitors) and re-suspend cells.
  4. Lyse germlings by gentle sonication (output 3.5; duty cycle 80; 1 second pulses) for 30 pulses and allow samples to sit on ice for 3 minutes.
  5. Centrifuge for 5 min at 14000rpm and 40C.
  6. Carefully pipette off and discard the supernatants of both aliquots
  7. Re-suspend each pellet in 500uL of ice cold NPS buffer with Calcium Chloride (after adding PMSF and Protease Inhibitors).
  8. Transfer both aliquots of each re-suspended sample into one new clean 15mL screw top falcon tube and store on ice.
  9. *Optional* If necessary wash tubes with another 500uL of NPS buffer with Calcium Chloride (after adding PMSF and Protease Inhibitors) and re-suspend any remaining sample.
  10. *Optional* Transfer both washes to the 15mL screw top falcon tube containing the combined aliquots.

Note: You MUST rinse the sonicator tip between samples to avoid cross contamination. First, rinse the tip liberally with 20% EtOH followed by a liberal rinse with dH2O, then carefully dry the tip with a Kimwipe.


MNase Digestion

  1. Bring volume of samples to 6mL with NPS buffer with Calcium Chloride (after adding PMSF and Protease Inhibitors) to each sample.
  2. Mix samples by pipetting up and down repeatedly.
  3. Pipette sample into epi-tubes in 700uL aliquots (should make 8).
  4. Add 0.1uL of Takara MNase amount to each aliquot and incubate in an 370C shaker for the indicated time interval (see *Example below).

*Example: If using Takara MNase (20 Units/uL) set digestion times to 5min, 10min, 20min, 40min, 60min, 90min, and 120min.

Note: Make sure to leave one aliquot of each sonicated sample undigested (w/ out MNase) as a negative control (effectively 0min).

Quenching MNase Digestion

  1. After each aliquots allotted digestion time has passed, add 15uL of 500mM EDTA (to inactivate MNase) and 25uL of 4M NaCl to quench the MNase Digestion.
  2. Pipette 20uL of each sample into a new 1.5mL epi-tube, label, and store in the -200C freezer (Input sample).
  3. Incubate remaining samples overnight @ 650C (to reverse cross linking).

Equilibration of protein A/G coupled Agarose Beads

  1. Equilibrate protein A/G-coupled agarose beads by washing in 10 volumes of ChIP lysis buffer (after adding PMSF and Protease Inhibitor).
  2. Centrifuge beads at 5000 RPM for 5min and then pipette off and discard the supernatant.
  3. Repeat steps 1 and 2.
  4. Return A/G beads to their original volume with ChIP lysis buffer, re-supsend A/G beads, and store on ice till used.


Note: Due to the size of the opening in the yellow 1uL-200uL pipette tips (CORNING), you must trim them when working with the A/G beads. First trim a template tip, then trim all tips used to this length to ensure consistency. Be sure to note that your volumes will be slightly higher than measured as a result of this alteration.


Overnight Incubation of Samples

  1. Carefully pipette each Sheared Chromatin Sample into a number of aliquots equal to one more than the number of antibodies (#AB +1) to be used in new 1.5mL epi-tubes.
  2. Be sure to mark down the volumes of your aliquots as this will be needed for later dilution calculations
  3. Label the first aliquot of each sample "No Antibody" and add 20uL of equilibrated A/G beads and 1uL of dH20.
  4. To each additional aliquot add 20uL of equilibrated A/G beads and 1uL of the desired antibody.
  5. Seal all samples with parafilm and incubate all sample tubes overnight at 40C on a rotator

Note: Due to the size of the opening in the pipette tips, you must trim them when working with the A/G beads (see above).

Note: Cell pellets in this protocol will be very loose, so be careful when pipetting. If needed, double centrifuge time to tighten pellets.

Note: Equilibrated beads can be stored at 40C for 1-2 days before use.

Note: We have tested protein G-coupled magnetic beads (dynabeads, invitrogen) and these yielded comparable results.


Day 2

Cold Washes

  1. Retrieve samples from 40C rotator and place on ice.
  2. Centrifuge samples for 1 min at 5000 RPM to pellet beads then pipette off and discard the supernatant.
  3. Wash Samples by adding 1mL of Ice Cold ChIP lysis buffer (w/o protease inhibitors) and incubate for 10 min at 40C on a rotating platform.
  4. Centrifuge Samples for 1 min at 5000 RPM to pellet beads then pipette off and discard the supernatant.
  5. Wash (as in steps 3-4) with Ice Cold ChIP lysis buffer (w/o protease inhibitors) one additional time.
  6. Wash (as in steps 3-4) with Ice Cold ChIP lysis buffer + 0.5M NaCl.
  7. Wash (as in steps 3-4) with Ice Cold LiCl Wash buffer.
  8. Wash (as in steps 3-4) with Ice Cold TE Buffer.


Note: All Wash steps above should be done on ice or in a 40C cold room.


Precipitation of Antibodies from A/G beads

  1. Centrifuge Samples for 2 min at 5000 RPM to pellet beads then pipette off and discard the supernatant (if not done at the end of previous step).
  2. To each sample pellet add 62.5uL of TES buffer and incubate in a 650C heat block for 10 mine to elute antibodies from A/G beads. Be sure to mix tubes by inversion several times during incubation.
  3. Centrifuge samples for 1 min at 5000 RPM to pellet beads and pipette off and SAVE the supernatant in a new 1.5mL epi-tube.
  4. Repeat steps 1 and 2 above, combining the supernatant in the same 1.5mL epi-tube as used above.


Note: Make sure not to disturb the A/G bead pellet, if necessary leave some of the supernatant behind to avoid this.


Preparation of Input Samples

  1. Remove Input Samples (see day 1) from -200C Freezer and thaw on ice.
  2. To Input Samples add 105 uL of ChIP TES Buffer and mix by inversion.

De-crosslinking of Samples

  1. Place all samples and input fractions in a 650C incubator and allow them to incubate for 6-16 hours (overnight).


Note: All steps should be done on ice or in the cold room until Precipitation step


Day 3

Digestion of RNase and Proteins

  1. Retrieve samples from the 650C incubator.
  2. Add 6uL of 10mg/ml RNAse A.
  3. Incubate samples at 500C for 2 hours.
  4. Add 6uL of 10% SDS (to linearize and inactivate proteins) and 10uL of 10mg/mL Proteinase K (to digest proteins).
  5. Incubate samples at 650C for 2 hours.


Phenol/Chloroform Extraction

  1. Retrieve Samples from 650C Incubator.
  2. Add 650uL of Phenol/Chloroform/IAA (25:24:1) to each sample and mix.
  3. Centrifuge Samples for 10min at 14,000RPM to separate layers.
  4. Extract aqueous layers of Samples by pipette and SAVE in new 1.5mL epi-tubes.
  5. Add 650 uL of pure Chloroform to the aqueous layers of Samples isolated in the last step and mix.
  6. Centrifuge Samples for 10min at 14,000PRM to separate layers.
  7. Extract aqueous layers of Samples by pipette and SAVE in new 1.5mL epi-tubes.
  8. Aliquot each sample into two equal volumes(approximately 325uL each) in new 1.5mL epi-tubes.
  9. Add 1uL glycogen, 32.5 uL 3M Sodium Acetate pH 5.2, and 1124.5uL of 100% EtOH to each aliquot.
  10. Place Samples in the -200C freezer to precipitate overnight.


Day 4

Preparation of DNA Precipitate Samples

  1. Retrieve samples from -200C freezer and thaw on ice
  2. Centrifuge samples for 10min at 14,000RPM.
  3. Pipette off the supernatant and discard.
  4. Wash samples by adding 300uL of 70% Ethanol.
  5. Centrifuge samples for 5min at 14,000RPM.
  6. Pipette off the supernatant and discard.
  7. Dry precipitate in Speed Vac under medium heat for 5 min or longer as necessary.
  8. Re-suspend samples in 25uL of TE Buffer.
  9. Qubit samples for concentration (mark on tubes) and store samples in -200C freezer until needed.




Recipes

For Immediate Use

NPS Buffer (10mL)

9.6mL water
100.0uL 1M Tris-HCl, pH 7.5
100.0uL 5M NaCl
50.0uL 1M MgCl2
5.0uL 1M Spermidine
7.0uL Beta-MercaptoEthanol
10.0uL 1.0M CaCl2

ChIP Lysis Buffer (10mL)

7.887mL water
500.0uL 1M HEPES-KOH or NaOH, pH 7.5
0.350uL 4M NaCl
20.0uL 0.5M EDTA
1.0mL 10% Triton-X 100
100.0uL 10% Deoxycholate (DOC)
100.0uL 0.1 M PMSF (in isopropanol)
33.0uL 333X leupeptin
10.0uL 1000X pepstatin

Note: Do not add the PMSF or protease inhibitors until immediately before solution will be used.

Note: If working with antibodies to acetylated histones, use 250uL 2M Na-Butyrate; NaCl concentration should be adjusted to 90mM.


Stock Solution Preparation

ChIP Lysis Buffer Without Protease Inhibitors (200mL)

160.6mL water
10.0mL 1M HEPES, pH 7.5
7.0mL 4M NaCL
400.0uL 0.5M EDTA
20.0mL 10% Triton X-100
2.0mL 10% DOC

Lysis Buffer plus 0.5M NaCl (200mL)

142.6mL water
10.0mL 1M HEPES, pH 7.5
25.0mL 4M NaCl
400.0uL 0.5M EDTA
20.0mL 10% Triton X-100
2.0mL 10% DOC

ChIP LiCl Wash Buffer (200mL)

167.6mL water
2.0mL Tris-HCl pH8.0
10.0mL 5M LiCl
10.0mL 10% IGEPAL CA-630 (NP40)
10.0mL 10% DOC
400.0uL 0.5M EDTA

ChIP TES Buffer (50mL)

41.5mL water
2.5mL Tris-HCl, pH 8.0
1.0mL 0.5M EDTA
5.0mL 10% SDS


Note: Do not refrigerate ChIP TES Buffer, store at room temperature and place in a 550C water bath for at least 15 min prior to use.


Note: Sigma claims that DOC disrupts the interaction between the M2 FLAG antibody and the epitope; however, we have performed successful ChIP of FLAG tagged proteins using these buffers. One may want to remove DOC for ChIP with FLAG tagged proteins.




Modified from the Lewis Lab Chromatin Immunoprecipitation From Germinating Conidia protocol and the MNase Protocol by Michael Seymour, 2013

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