Silver: ChIP-chip

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Day 1-2 - Crosslinking/Pre-Incubation of Beads with Antibody

Crosslinking

1. Pick 5 colonies of each yeast strain and grow up in 10 ml YEPD overnight
2. Grow up 5 separate 50 ml cultures from the 10 ml cultures to an OD₆₀₀ of 0.5 to 1.0 in YEPD (50 µl from saturated 10 ml culture into 50 ml fresh YEPD for overnight growth yields the approximate OD range the next morning)
   • For alpha factor induction the OD₆₀₀ should be 0.2 before adding alpha factor
     • Add 1 µl of 2.5 mg/ml alpha factor (final concentration is 30 nM) for 2 h at 30°C
3. Add 1.4 ml of 37% formaldehyde for every 50 ml culture
4. Parafilm the flask and place on the shaker on which it was grown (usually 30°C). Let incubate with formaldehyde for 20 min
5. Transfer cells to 50 ml falcon tubes and pellet at 4°C for 5 min at 3,000 rpm
6. Pour off formaldehyde waste into formaldehyde waste bottle
7. Resuspend cells in 25 ml of 1x TBS (tris-buffered saline)
   • 5x TBS
     • 100 ml Tris-HCl pH 7.5
     • 150 ml 5M NaCl
     • 750 ml ddH₂O
     • Filter sterilize and store at 4°C
     • After diluting the 5x to 1x filter again
8. Repeat wash with 10 ml of 1x TBS. Waste can be dumped down the sink
9. Resuspend pellets in 1 ml of 1x TBS and transfer to 1.5 ml eppendorf tubes
10. Centrifuge quickly at RT to pellet, remove supernatant, and flash freeze in liquid nitrogen
11. Tubes/cells can be stored at -80°C until experiment time

Pre-Incubation of Beads with Antibody

1. Use 50 µl of magnetic beads per IP (Pan-mouse IgG product # 110.22 Dynal Dynabeads) and place in a 15 ml falcon tube. (I combine this step, so for 10 IP's I put 500 µl beads into one 15 ml tube, make sure beads are completely resuspended in stock bottle before removing)
2. Spin for 1 min at 3,000 rpm at 4°C in tabletop centrifuge
3. Use large magnet to attract beads and remove the supernatant with a pipette
4. Immediately replace liquid (before beads dry) with 15 ml of 1x PBS with 5 mg/ml BSA (cold and filtered, made fresh).
   • 400 ml 1x PBS/BSA
     • 40 ml 10x PBS
     • 360 ml ddH₂O
     • 2 g BSA
5. Spin again, aspirate, and repeat wash with another 15 ml of 1x PBS/BSA
6. Spin again, aspirate, and resuspend beads in 500 µl of 1x PBS/BSA for 10 IPs (depending on # of IPs this amount varies, just 50 µl per IP)
7. For one IP, transfer 50 µl bead solution into a 0.5 ml tube and add 250 µl 1x PBS/BSA along with 10 µl of 9E11 monoclonal anti c-myc antibody
8. Parafilm tubes and then place them in a 1.5 ml Eppendorf tube, tape them in place. Place the tubes on a rotator in the cold room (4°C) overnight

Day 3 - Cell Lysis/Sonication/IP

Cell lysis

1. Make fresh lysis buffer (Need ~2.5 ml lysis buffer for each sample, 45ml for ~18 samples)
   • For 45 ml of 1x lysis buffer
     • 4.5 ml 0.5 M HEPES-KOH pH7.5
     • 1.26 ml 5M NaCl
     • 4.5 ml 10% Triton X-100
     • 90 µl 0.5 M EDTA
     • 450 µl 10% Na-deoxycholate
     • 45 µl 1000x PLAAC (protease inhibitor cocktail)
     • 225 µl 200 mM PMSF (made fresh, 150 mg PMSF + 4.3 ml 100% EtOH)
     • 34 ml ddH₂O
2. Thaw crosslinked cell pellets on ice
3. Resuspend the pellets in 200 µl lysis buffer and transfer to 1.5 ml screwcap tube
4. Add micro glass beads to just under the level of the liquid
5. Fastprep 1 x 40 sec, Power 6.5. Check under microscope, want >95% lysis, if not do 15-30 sec more
6. Add an additional 500 µl of lysis buffer to each lysate and vortex briefly
7. Pierce bottom of tube using 18 x 1 1/2 G needle. Place in a 2 ml screwcap tube and parafilm the two tubes together. Make sure hole is very tiny as any beads that go through will damage the sonicator tip later
8. Place tubes in 4°C centrifuge without lid and pulse up to 7,000 rpm then stop
9. Remove the 1.5 ml screwcap tube from the top, place caps on the 2.0 ml tubes, and pellet the insoluble chromatin (ie, the material we want) at 14,000 rpm for 7 min at 4°C
10. Remove the soluble portion of the lysate and discard (do not want competition from soluble protein)
11. Resuspend pellet in ~500 µl of lysis buffer, transfer to a 2.0 ml NUNC tube for sonication

Sonication

1. Bring up volume to 1.8 ml with lysis buffer (using markings on side of the tube is sufficient)
2. Immediately before sonicating each pellet, make sure it is in suspension. Pellets are sonicated 5 x 20 sec pulses at Power 4.0 with 30 s rests on ice in between pulses. Make sure the sonicator is tuned
3. Transfer contents of NUNC tubes to 2.0 ml snap-cap tubes and spin for 1 min at 4°C at 14,000 rpm
4. Transfer soluble portion (which can now be labeled as whole cell extract - WCE) to a new tube, let sit on ice as you wash beads from the prior day

IP

1. Spin the 0.5 ml tubes in 1.5 ml Eppendorfs at 3,000 rpm for 1 min at 4°C
2. Make fresh 1x PBS/BSA (5mg/ml)--filter
3. Use small magnet, suck off liquid and replace with 500 µl of 1x PBS/BSA. Transfer to a 15 ml conical
4. Add 10 ml 1x PBS/BSA to the 15 ml conicals and then place on rotator for 15 min at 4°C
5. Centrifuge at 3,000 rpm for 1 min at 4°C in table-top centrifuge
6. Using magnet, suck off 10 ml of 1x PBS/BSA and replace with fresh 1x PBS/BSA
7. Repeat 15 min incubation at 4°C
8. Spin down and resuspend Dynabeads in 200 µl lysis buffer (made fresh for the 2nd time that day because of decreased protease inhibitor half-life in aqueous solution)
9. Transfer 200 µl Dynabeads (in lysis buffer) to a 2 ml Eppendorf tube along with 1.2 ml of WCE and incubate on rotator at 4°C overnight. Save remaining WCE at - 20°C for use in future steps

Day 4 - Post-IP Washes/Elution/Reversing Crosslinks

Post-IP Washes

1. Make fresh lysis buffer (see above) and 200 mM PMSF (150 mg + 4.3 ml 100% EtOH)
2. Make fresh high salt lysis buffer (360 mM NaCl final)
   • 3.0 ml0.5M HEPES-KOH pH7.5
   • 2.16 ml 5 M NaCl
   • 3.0 ml 10% Triton X-100
   • 60 µl 0.5M EDTA
   • 300 µl 10% Na-deoxycholate
   • 30 µl PLAAC
   • 150 µl 200 mM PMSF
   • 21.3 ml ddH₂O
3. Spin down Dynabeads/lysate at 3,000 rpm for 1 min at 4°C
4. Use small magnet, save supernatant and replace with 1 ml cold lysis (not high salt) buffer (you will quantify the protein levels in the WCEs, and in the depleted supernatants, and will run it on a gel along with some of the IP eluate to determine if your IP worked)
5. Incubate beads in lysis buffer for 5 min on rotator at 4°C
6. Repeat the 1 ml lysis buffer wash at 4°C
7. Wash 2x 1 ml "high salt” lysis buffer at 4°C
8. Wash 2x 1 ml "wash" buffer at 4°C
   • 5 ml 1 M Tris-HCl pH 8.0 (10 mM Tris-HCl pH 8.0)
   • 25 ml 5 M LiCl (250 mM LiCl)
   • 2.5 ml 100% NP40 (0.5% NP40)
   • 25 ml 10% Na-deoxycholate (0.5% Na-deoxycholate)
   • 1 ml 0.5M EDTA (1 mM EDTA)
   • 441.5 ml ddH₂O
     • Filter and store in the cold room
     • 5 M LiCl = 21.2g LiCl up to 100 ml ddH₂O

Elution

1. To the rest of the samples, add 50 µl elution buffer
   • 5 ml 1 M Tris-HCl pH 8.0 (50 mM Tris-HCl pH 8.0)
   • 2 ml 0.5 M EDTA (10 mM EDTA)
   • 10 ml 10% SDS (1% SDS)
   • 83 ml ddH₂O
     • Filter and store at RT
2. Vortex briefly before beginning incubation at 65°C for 10-15 minutes. Vortex briefly every 2 min during the incubation. You can't overdo this step, so if you want to let it go 20 min then that's just fine
3. Spin tubes at RT at 14,000 rpm for 30 seconds

Reversing Crosslinks

1. Using magnet to hold beads, transfer 30 µl of IP samples to a new tube and add 170 µl of TE/SDS
   • 1 ml Tris-HCl pH 8.0 (10 mM Tris-HCl pH 8.0)
   • 200 µl 0.5 M EDTA (1 mM EDTA)
   • 1.6 ml 10% SDS (1 % SDS)
   • 98 ml ddH₂O
     • Filter and store at RT
2. Since you do not use all of the IP, some will be available for checking by Western
3. Thaw WCE from previous day, add 5 µl to a new tube and add 195 µl TE/SDS
4. Incubate tubes at 65°C overnight to de-crosslink, use a heat-block or an adjustable water bath. Not a bad idea to vortex and spin down the tubes once before you go home, just to make sure everything is being mixed and de-crosslinked

Day 5 - Proteinase K Digestion/PCI Extraction and DNA Precipitation

Proteinase K Digestion

1. Remove tubes from 65°C and prepare Proteinase K mix--let tubes return to RT before adding Proteinase K mix as you do not want to heat-denature your Proteinase K
2. Proteinase K Mix (per sample)
   • 186.67 µl TE
   • 4 µl glycogen (20 mg/ml)
   • 10 µl Proteinase K (20 mg/ml)
     • May need to make this up in 15 ml conical since you have twice as many samples now (IP + WCE) to deal with
3. Add 200 µl of Proteinase K mix to each sample and incubate tubes for 2 hours at 37°C

PCI Extraction and DNA Precipitation

1. Transfer samples (~400 µl) to 2.0 ml Phase-Lock gel tubes (Eppendorf), tubes should be flash-spun before use just to get the gel down to the bottom of the tube
2. Add 400 µl of cold phenol:chloroform:isoamyl alcohol (PCI) to each sample and pipette to mix. Do NOT vortex
3. Spin at 13,000 rpm for 5 min at RT
4. Transfer aqueous (~370 µl, everything above the gel layer which defines the boundary between aqueous and phenol) to a 1.5 ml Eppendorf tube
5. Add 14.8 µl 5M NaCl to each tube (gives ~200 mM NaCl final concentration)
6. Add 2 volumes (~740 µl) of cold 100% EtOH, vortex briefly, and incubate at -20°C for at least 2 days

Day 6 - RNase A Treatment/Blunting/PCI Extraction and DNA Precipitation/Preparation of Linkers

RNase A Treatment

1. Spin at 14,000 rpm for 40 min at 4°C (cold room-should see fluffy white pellet since there's still RNA around)
2. Carefully pipette off supernatant and replace with 1 ml cold 70% EtOH, vortex briefly (setting 5), and spin at 14,000 rpm for 15 min at 4°C. It is not necessary to remove all of the ethanol if you feel you will disturb the DNA pellet or the pellet is loose
3. Carefully pipette off supernatant, spin briefly and remove the remaining liquid with a pipette
4. Air-dry for a long time (2-3 h). It is absolutely essential that the tubes be completely dry-the pellet should become somewhat clear and glassy looking
5. Resuspend pellet in 30 µl of TE (pH 8.0). To get the DNA fully into solution, I've found that incubating the tubes at 50°C with frequent vortexing (setting 5) and quick flashspins help it to go into solution. I do this for about 10 to 15 min (the DNA survives 65°C overnight so the 50°C waterbath is NOT going to hurt it)
6. Add 1 µl of preboiled 10 mg/ml RNAse A to each tube and incubate at 37°C for 1 h
7. Purify using Qiagen gel purification/PCR cleanup kit and elute with 50 µl of EB Buffer (10mM Tris pH 8.0). Have ~48 µl left here. Add the 50 µl back to the filter and spin an extra time to recover as much DNA as possible

Blunting

1. In separate PCR tubes, combine 2 µl of WCE + 38 µl ddH₂O. For the IPs, add 40 µl of immunoprecipitated DNA (no H₂O needed)
2. To each, add 70 µl of
   • 11 µl 10x T4 DNA pol buffer
   • 0.5 µl 10mg/ml BSA
   • 0.5 µl dNTP mix (20 mM each, made fresh immediately before use)
   • 0.2 µl T4 DNA pol (3U/µl)
   • 57.8 µl ddH₂O
3. Mix by pipetting and incubate at 12°C for 20 min in a PCR block. Do not use the heated lid option

PCI Extraction and DNA Precipitation

1. Place on ice and add 12 µl of this mix
   • 11.5 µl 3M NaOAc pH 5.2
   • 0.5 µl glycogen (20mg/ml)
2. Mix by vortexing and transfer to a 0.5 ml Phase-Lock gel extraction tube. Add 120 µl of PCI and mix by pipetting
3. Spin for 5 min at 13,000 rpm, RT
4. Transfer ~110 µl to a new 1.5 ml tube and add 230 µl ice cold 100% EtOH
5. Incubate at -20°C for at least 2 h (The longer the better, shoot for two days)
6. Order Fresh Cy3-dUTP and Cy5-dUTP

Preparation of Linkers

• Best done the day before you need them

1. Need 6.7 µl mix per sample
   • 30 µl of 40 µM oJW102 (5'--GCG GTG ACC CGG GAG ATC TGA ATT C--3')
   • 30 µl of 40 µM oJW103 (5'--GAA TTC AGA TC--3')
   • 20 µl of 6x SSC
2. Start tubes at 95°C in water bath. Let water and tubes cool to room temperature, then place at 4°C overnight for use with ligations the next day

Day 6 - Ligation

1. Spin for 30 min at 14,000 rpm at 4°C
2. On ice, pipette off supernatant and wash the pellet with 500 µl cold 70% EtOH
3. Spin for 20 min at 14,000 rpm at 4°C
4. On ice, pipette off supernatant, flash spin and remove any remaining liquid. Cover and allow to air dry at room temperature for 2 h
5. Resuspend pellet in 25 µl ddH₂O (use the 50°C water bath with vortexing again to help it go into solution, 10-15 min at least). Once the pellet is in solution place the tubes on ice
6. To setup ligations
   • Add 25 µl of cold ligase mix to each tube (make a stock solution, 5x or 6x for 4 samples)
     • 9.67 µl ddH₂O
     • 5 µl 10x DNA Ligase Buffer
     • 6.7 µl Linkers (mix of 40 µM each, from previous day)
     • 0.5 µl T4 DNA Ligase
     • 3.33 µl 15 mM ATP
7. Mix by pipetting and spin down to make sure all the liquid is at the bottom and incubate at 16°C
   • Let this go for at least 2 days

Day 7 - DNA Precipitation/PCR Amplification

DNA Precipitation

1. Add 6 µl of 3M NaOAc (pH 5.2) and 130 µl of cold 100% EtOH, mix by vortexing and spin for 25 min at 4°C at 14,000 rpm (DO NOT incubate at -20°C here—do not want to precipitate un-ligated linkers which will ruin the PCR)
2. Pipette off supernatant and wash with 500 µl 75% EtOH
3. Spin for 10 min at 4°C
4. Pipette off supernatant, spin and remove remaining liquid with a pipette, air dry at RT for 2h

PCR Amplification

1. Resuspend pellets in 25 µl ddH₂O and place on ice (50°C, vortexing as before), transfer to PCR tubes
2. Add 25 ul of PCR mix to each tube:
   • 5 µl 10x Thermopol buffer
   • 11.75 µl ddH₂O
   • 5 µl dNTP (2.5 mM each of all 4)
   • 1.25 µl oligo oJW102 (40 µM stock)
   • 1 µl Amplitaq polymerase (5 U/µl)
   • 1 µl Pfu Turbo dilution (dilute 1 µl of Pfu Turbo, 2.5 U/µl, in 20 µl ddH₂O)
3. Place samples in the PCR machine with the following program (takes ~1.5 h)

Step Time Temp 1 8 min 55°C (make longer if a lot of samples) 2 5 min 72°C 3 2 min 95°C 4 30 sec 95°C 5 30 sec 55°C 6 1 min 72°C 7 go to step 4 for 24x 8 4 min 72°C 9 infinity 10°C

1. After PCR amplification, check product on gel: 5 µl DNA, 5 µl H₂O, 2 µl 6x DNA dye, no Bromphenol Blue (runs at same level as sonicated DNA), run with 1 kb and 100 bp ladders (1.2% agarose gel). Run for 30 min at 100V, look for a smear around 350-400bp
2. Use Qiagen PCR purification kit and elute in 65 µl of their elution buffer

Day 8 - Klenow-Mediated Incorporation of Dyes/DNA Precipitation

Klenow-Mediated Incorporation of Dyes

• Cy5-dUTP = IP = blue
• Cy3-dUTP = WCE = pink

1. Transfer 21 µl of DNA to an eppendorf tube
2. Add 20 µl of 2.5 x random primer solution (from BioPrime kit) to tube
3. Boil for 5 min at 100°C, then put on ice, incubate for 5 min
4. Add 5 µl of 10x dNTP (low T mix)
   • Low T dNTP Mix
     • 1.2 µl dATP
     • 1.2 µl dCTP
     • 1.2 µl dGTP
     • 0.6 µl dTTP
     • 95.8 µl ddH₂0
5. Add 3 µl of Cy5-dUTP to IP sample, and 3 µl of Cy3-dUTP to WCE sample, do in the dark since the dyes are light sensitive
6. Add 1 µl of high concentration Klenow (from BioPrime kit), make sure to mix well
7. Incubate at 37°C for 1 h (wrap in aluminum foil to keep dark)
8. Use Qiagen purification kit to purify the DNA, elute in some considerable amount of EB buffer from kit (100 µl is fine-just want to get as much off the column as possible and the volume change doesn't matter since you'll be precipitating anyway)

DNA Precipitation

1. Add 12 µl of 3M NaOAc, 260 µl of 100% EtOH. Incubate at -20°C for at least a couple of days (wrap in aluminum foil to keep dark)
2. Spin at 14k rpm, 4°C, 40 min
3. Wash with 500 µl 75% EtOH
4. Spin at 14k rpm, 4°C, 20 min
5. Air dry for at least 2 h. Should have nice, tight, bright pellets