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| ==Protocol== | | ==Protocol== |
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| Buffers:-
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| IP Buffer
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| 50 mM HEPES pH to 7.5 with KOH
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| 150 mM NaCl
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| 1 mM EDTA
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| 1 % Triton X-100
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| 0.1 % Sodium deoxycholate
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| 0.1 % SDS
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| 5X Elution Buffer
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| TE pH 7.6
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| 5% SDS
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| IP Buffer + salt
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| 50 mM HEPES pH to 7.5 with KOH
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| 500 mM NaCl
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| 1 mM EDTA
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| 1 % Triton X-100
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| 0.1 % Sodium deoxycholate
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| 0.1 % SDS
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| Wash Buffer
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| 10 mM Tris pH 8.0
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| 250 mM LiCl
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| 1 mM EDTA
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| 0.5 % Nonidet P-40 substitute
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| 0.5 % Sodium deoxycholate
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| 1X Elution Buffer
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| TE pH 7.6
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| 1 % SDS
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| Pre-germination:
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| 1) Normalise the spores, for multiple samples
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| 2) Add the spores to 1 ml TES buffer, centrifuge and remove the supernatant, re-suspend in 1 ml TES buffer
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| 3) Heat shock at 50˚C for 10 minutes
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| 4) Add spores to 5 ml 2X PG media + 10 µl 5 M CaCl2 and leave at 37˚C for 3 hours
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| 5) Vortex vigorously
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| 6) Add spores to media, typically 3 ml into 2 flasks of 80 ml YEME (10% glucose) + 160 µl MgCl2 and 80 µl 10% antifoam
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| 7) Leave shaking overnight at 300 rpm, 30˚C
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| 8) Wait until the cultures are at OD600 = 0.8-1
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| Cross-linking:
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| 9) Add 1% formaldehyde and place back in 30˚C, 300 rpm for 20 min
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| 10) Add 0.05 M glycine and incubate for a further 5 min
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| 11) Harvest cells by centrifugation @ 6000 rpm for 5 min @ 4˚C
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| 12) Wash cells twice with 25 ml ice-cold PBS (pH 7.4)
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| 13) Store pellet @ -80˚C
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| Chromatin extraction:
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| 1) Label the bead beater cups (and corresponding lids) with marker pen & place in -80°C freezer for 1-2 hrs minimum before the extraction.
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| 2) Add a frozen cell pellet straight from the -80°C freezer to each cup (two cups): Add the ball-bearing on top of the frozen pellet and screw on the lid & immerse in Liquid Nitrogen.
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| 3) Place both cups (need 2 for balance) in machine and grind for 1 min 30 sec at a frequency of 30 Hz (maximum setting). You should hear a loud noise with a lot of fast metallic “clicking” if the ball-bearing is moving freely.
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| 4) Repeat the cooling & grinding steps again for a total of 3 rounds. The pellet should have been reduced to a fine, friable, dry looking powder without any lumps. Place one of the cups in the - 80°C freezer to keep it cold whilst you process the other cup.
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| 4) Remove lid, and remove ball bearing using a magnet (scrapping off powder from ball bearing if necessary). Scrape powder into a 15 ml centrifuge tube.
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| Extracting Chromatin:
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| 1) Add 2.2 ml IP buffer (to a pellet from a 40 ml initial culture) containing RNAse A (0.1 mg/ml) + Protease Inhibitor tablet
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| 2) Divide each 15 ml tube into 6 x 400 µl aliquots & sonicate using a Biorupter for 35 cycles with 30s ON & 30s OFF.
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| 3) Spin all samples at 16,000 x g for 15 min.
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| 4) Transfer supernatant to new tubes (6 x 400 µl aliquots]. This is your chromatin prep
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| 5) Take 14 µl of each sample + 4 µl of 5 x Elution Buffer (TE pH 7.6, 5% SDS) + 1 µl of RNAse (stock 1 mg/ml) & leave at RT for 30 min. Put all of this in a PCR tube.
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| 6) Freeze remainder of supernatant (380l) at -20⁰C.
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| 7) Add 2 µl of Proteinase K (stock: 10 mg/ml) & leave at 42⁰C for 2 hrs.
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| 8) De-cross link at 65⁰C for ~ 6 hrs (overnight in PCR machine).
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| 9) Next day run 5 μl aliquots on a gel with orange loading dye on 1.2% agarose gel to check fragment sizes.
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| 10) Check sample fragment sizes most closely matche the ideal size range of between 50-500 bp for future work. Sonicate further if needed.
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| Chromatin Immunoprecipitation:
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| 1) Take samples from freezer and place in 2 ml Eppendorf tube. Pre-clear the chromatin samples by adding 25 μl of NEB Protein G or Protein A magnetic beads depending on what antibodies will be used.
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| 2) Incubate the chromatin and beads at 4°C (cold room) for 1.5 hour in a rotator set to 20 rpm. This step allows any material in the chromatin prep which non-specifically adheres to the beads to bind and be removed prior to the immunoprecipitation
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| 3) After the incubation immobilise the beads using a magnetic rack and transfer the liquid (cleared chromatin) to a fresh labelled tube.
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| 4) Take 100 μl of pre-cleared chromatin (input control sample) & freeze at -20⁰C until IP are ready for de-crosslinking.
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| 5) To remaining chromatin material add the antibody to your protein of interest (1-5 μg) to the cleared chromatin. Use 7 μl of the antibody (stock: 1mg/ml) and incubate at 2.5 hrs at 4°C (cold room) in a rotator set to 20 rpm.
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| 6) Add 30 μl of Protein G or A magnetic beads to the chromatin and antibody and leave for 1.5 hr in the 4°C (cold room) in a rotator set to 20 rpm.
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| 7) Immobilise the beads using a magnetic rack and remove (and keep) the spent chromatin liquid
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| 8) Re-suspend the beads by gentle pipetting (do not vortex) in 750 μl of ice-cold IP buffer. Transfer the re-suspended beads to a new non-stick 1.5 ml tube (Ambion/Fisher cat no VYAM12450) and do the washes.
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| 9) Washes: incubate the re-suspended beads in IP Buffer at 4°C (cold room) for 10 min in a rotator set to 20 rpm. Then immobilise the beads using a magnetic rack and discard the IP Buffer.
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| 10) Re-suspend the beads in 1 ml of ice-cold IP Buffer + Salt and again incubate at 4°C (cold room) for 10 min in a rotator set to 20 rpm. Again immobilise the beads using a magnetic rack and discard the IP Buffer + Salt.
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| 11) Re-suspend the beads in 1 ml of ice-cold IP Wash Buffer and again incubate at 4°C (cold room) for 10 min in a rotator set to 20 rpm. Immobilise the beads using a magnetic rack and discard the IP Wash Buffer .
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| 12) Re-suspend the beads in 1 ml of ice-cold TE pH 8 and again incubate at 4°C (cold room) for 10 min in a rotator set to 20 rpm. Immobilise the beads using a magnetic rack and discard the TE pH 8. Repeat this step.
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| 13) Re-suspend the beads in 100 μl of 1 x Elution Buffer and add 5 μl of RNAse A (stock: 1mg/ml) & leave @ RT for 30min.
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| 14) Take out the 100 μl of input control chromatin & add 5 μl of RNAse A (stock: 1mg/ml) & leave @ RT for 30min.
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| 15) To the IP & input: de-crosslink by heating to 65°C waterbath overnight.
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| 16) Next day, Immobilise the beads using a magnetic rack and transfer the liquid which contains the immunoprecipitated DNA to a fresh labelled tube.
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| 17) Re-suspend the beads in 50 μl of TE pH 7.5 to wash any remaining DNA from them. Immobilise the beads using a magnetic rack and transfer the 50 μl of TE pH 7.5 to the other immunoprecipitated DNA solution.
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| 18) Add 50 μl TE to the total input control DNA sample so that all samples are 150 μl.
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| 19) Add 5 μl of 10 mg/ml Proteinase K & incubate at 55°C for 2 hrs in the waterbath. This step destroys the proteins.
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| 20) To Both IP & input:
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| -Pass the samples through a PCR purification column (Qiagen kit – MiniElute Kit is recommended).
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| -Nanodrop 1.5 μl.
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