MNase Protocol w/ out Cross-linking

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This is an alternative to the standard MNase Protocol which does not utilize cross-linking of proteins to DNA. It has not yet been tested

Contents

Pre-Preparation

Preparation of Solid Culture Media

  1. Prepare one 50mL aliquot of 1.5% Sucrose, 1x Vogel's liquid media for each strain to be used in the experiment.
  2. Prepare one 250mL Erlenmeyer Flasks for each strain by adding 0.75g of Agar (final concentration will be 1.5% in 50mL)
  3. Pipette a 50mL aliquot of liquid media into each Flask loaded with agar and cap with a foam plug.
  4. Autoclave media and mix by swirling before allowing it to cool to room temperature on bench.

Note: When preparing media, allow it to cool to room temp after autoclaving before use.

Note: Heat sensitive media components, such as Ethanol, can be added after autoclaving from a sterile source before the media solidifies.


Preparation of Liquid Culture Media

  1. Prepare 50mL of 2% Glucose, 1x Vogel's liquid media for each media/strain combination to be used and add additional media components as necessary.
  2. Pipette liquid media into 125mL Erlenmeyer Flasks in 50mL aliquots and cap with foam plugs.
  3. Autoclave media and allow to cool to room temperature and store on bench.

Note: When preparing media, allow it to cool to room temp after autoclaving before use.

Note: Heat sensitive media components, such as Ethanol, can be added after autoclaving from a sterile source before the media solidifies.


Inoculation of Stock Cultures

  1. 6 days before the start of the experiment, inoculate 2 flasks of Solid Culture media with each Strain to be used in the experiment utilizing sterile technique.
  2. Grow cultures in 320C incubator for 72 hours
  3. Remove cultures from incubator and store on bench @ room temp until needed (best if used within two weeks)


Preparation of Reagents

  1. Prepare all reagents as needed (see Recipes section below).


Day 1

Preparation of Conidial Suspension

  1. Add 50mL of Sterile H2O to the desired stock culture (at least 6 days of growth).
  2. Vortex culture vigorously until conidia on glass and agar come loose and go into suspension.
  3. Strain resulting supernatant through sterile cheesecloth to remove Agar and Mycelia.
  4. Pour resulting conidial suspension into a 50mL Falcon tube and centrifuge @ 3000 RPM for 10 min in swinging bucket centrifuge.
  5. Carefully pipette off the supernatant and discard.
  6. Re-suspend conidia by bringing volume up to 5mL with dH2O and vortexing.


Inoculation of Liquid Cultures

  1. Inoculate one of each type of liquid culture media to be used in the experiment with approximately 1x106 conidia/mL of each strain to be used in the experiment. Depending on concentration of the conidial suspension used, the inoculum should be approximately 125uL.
  2. Incubate inoculated liquid cultures for 4-5 hours with shaking (200rpm) at 320C
  3. Check for germination under 100x magnification in a light microscope

Example: If your are using 3 types of liquid media (2%Glucose, 2%Ethenol, and 2%Glucose + 2% Ethenol) and two strains(WT and Mutant-X), inoculating one flask of each type of media with each strain would result in a total of 6 inoculated liquid cultures.


Harvesting Germlings

  1. Transfer liquid cultures to 50ml Screw Top Falcon Tubes and centrifuge at 3000 RPM for 10min to pellet the cells.
  2. Carefully pipette off the supernatant and discard.
  3. Add 40mL of 1xPBS buffer and re-suspend the cells.
  4. Centrifuge at 3000 RPM for 10 min to pellet cells.
  5. Carefully pipette off and discard the supernatant.
  6. Add 10 mL of 1xPBS 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 ChIP lysis buffer (after adding PMSF and Protease Inhibitors) and re-suspend cells.
  4. Lyse mycelia by sonicating (output 3.5; duty cycle 80; 1 second pulses) for 30 pulses and allow samples to sit on ice for 3 minutes.
  5. Shear chromatin by sonicating samples for 20 pulses (output 3.5; duty cycle 80; 1 second pulses) followed by 3 minutes on ice.
  6. Repeat above step five (5X) more times (for a total of 6 cycles of 20 pulses).
  7. Centrifuge for 5 min at 14000rpm and 40C.
  8. Carefully pipete off and recombine the supernatants of both aliquotes of each sample into one new clean 15mL screw top falcon tube and store on ice.
  9. Pipette 20uL of each recombined sample into a new 1.5mL epi-tube, label, and store in the -200C freezer (Input sample).

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.


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 aliquotes equal to one more than the number of antibodies (#AB +1) to be used in new 1.5mL epi-tubes.
  2. Label the first aliqote of each sample "No Antibody" and add 20uL of equilibrated A/G beads and 1uL of dH20.
  3. To each additional aliquote add 20uL of equilibrated A/G beads and 1uL of the desired antibody.
  4. 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. Retreive 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 ChIP lysis buffer (w/o protease inhibitors) one additional time.
  6. Wash (as in steps 3-4) with ChIP lysis buffer + 0.5M NaCl.
  7. Wash (as in steps 3-4) with LiCl Wash buffer.
  8. Wash (as in steps 3-4) with 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. 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.
  2. 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.
  3. 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 2) from -200C Freezer and thaw on ice.
  2. To Input Samples add 105 uL of ChIP TES Buffer and mix by inversion.


Precipitation of Chromatin

  1. To each sample add 125 uL water and 2.5 ul 10mg/ml RNAse A.
  2. Incubate for 2h at 500C.
  3. Add 6.25 uL 20mg/mL Proteinase K to each sample.
  4. Incubate samples for 2h at 500C.
  5. Add 250 uL Phenol/Chloroform/IAA (25:24:1) to each sample and mix.
  6. Centrifuge Samples for 10min at 14,000RPM to separate layers.
  7. Extract aqueous layers of Samples by pipette and SAVE in new 1.5mL epi-tubes.
  8. Add 250 uL of pure Chloroform to the aqueous layers of Samples isolated in the last step and mix.
  9. Centrifuge Samples for 10min at 14,000PRM to separate layers.
  10. Extract aqueous layers of Samples by pipette and SAVE in new 1.5mL epi-tubes.
  11. Add 1uL glycogen, 25 uL 3M Sodium Acetate pH 5.2, and 865 uL 100% EtOH to the aqueous layers of Samples isolated in the last step.
  12. Place Samples in the -200C freezer to precipitate overnight.


Day 3

Preparation of Chromatin 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. To Wash samples add 300 uL 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 5min or longer as necessary.
  8. Re-suspend samples in 25uL of TE Buffer.
  9. Store in -200C freezer until needed.


Perform multiplex or RealTime PCR

See associated protocols



Recipes



ChIP Lysis Buffer (10mL)

7.88mL 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)
10.0uL 1000X leupeptin
10.0uL 1000X E-64
33.0uL 300X 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.

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 MNase Protocol protocol by Michael Seymour, 2013

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