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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 ul 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 OD should be 0.2 before adding - Add 1ul of 2.5mg/ml alpha factor (final concentration is 30 nM) for 2 h at 30°C 3. Add 1.4 mls of 37% formaldehyde for every 50 ml culture. Formaldehyde is under the chemical hood in Rm 924. 4. Parafilm the flask and replace 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 3000 rpm. 6. Pour off formaldehyde waste into formaldehyde waste bottle in hood in Rm 924. 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 room-temp to pellet, remove supernatant, and flash freeze in liquid nitrogen. 11. Tubes/cells saved at -80°C until experiment time.
Pre-incubation of beads with antibody - day before you sonicate:
1. Use 50 ul of 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 ul beads into one 15 ml tube, make sure beads are completely resuspended in stock bottle before removing) 2. Spin for 1 min at 3000 rpm at 4°C in tabletop centrifuge. 3. Use large magnet to attract beads and remove the supernatant with a pipette. 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 (4°C, common fridge in Rm 916)
4. Spin again, aspirate, and repeat wash with another 15 ml of 1x PBS/BSA. 5. Spin again, aspirate, and resuspend beads in 500 ul of 1x PBS/BSA for 10 IPs (depending on # of IPs this amount varies, just 50 ul per IP). 6. For one IP, transfer 50 ul bead solution into a 0.5 ml tube and add 250 ul 1x PBS/BSA along with 10 ul of 9E11 monoclonal anti c-myc antibody (common fridge, Rm 916). 7. 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.
Cell lysis, chromatin sonication, IP setup:
1. Make fresh lysis buffer (Need ~2.5 ml lysis buffer for each sample, 45ml for ~18 samples) 2. For 45 ml of 1x: 4.5 ml 0.5 M HEPES-KOH pH7.5 1.26 ml 5M NaCl 4.5 ml 10% Triton X-100 90 ul 0.5 M EDTA 450 ul 10% Na-deoxycholate 45 ul 1000x PLAAC 225 ul 200 mM PMSF (made fresh, 150 mg + 4.3 ml 100% EtOH) 34 ml ddH2O 3. Thaw crosslinked cell pellets on ice. 4. Resuspend the pellets in 200 ul lysis buffer and transfer to 1.5 ml screwcap tube (1415-8500). 5. Add micro glass beads to just under the level of the liquid. 6. Fastprep 1 x 40 sec, Power 6.5. Check with scope, want >95% lysis, if not do 15-30 sec more 7. Add an additional 500 ul of lysis buffer to each lysate and vortex briefly. 8. 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. 9. Place tubes in 4°C centrifuge without lid and pulse up to 7,000 rpm then stop. 10. 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. 11. Remove the soluble portion of the lysate and discard (do not want soluble protein competing) 12. Resuspend pellet in ~500 ul of lysis buffer, transfer to a 2.0 ml NUNC tube for sonication. 13. Bring up volume to 1.8 ml with lysis buffer (using markings on side of the tube is sufficient). 14. 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 before using. 15. Transfer contents of NUNC tubes to 2.0 ml snap-cap tubes and spin for 1 min at 4°C at 14,000 rpm 16. 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. 17. Spin the 0.5 ml tubes in 1.5 ml Eppendorfs at 3,000 rpm for 1 min at 4°C (cold room). 18. Make fresh 1x PBS/BSA (5mg/ml)--filter. 19. Use small magnet, suck off liquid and replace with 500 ul of 1x PBS/BSA. Transfer to a 15 ml conical. 20. Add 10 ml PBS/BSA to the 15 ml conicals and then place on rotator for 15 min at 4°C. 21. Centrifuge at 3,000 rpm for 1 min at 4°C in table-top centrifuge. 22. Using magnet, suck off 10 ml of 1x PBS/BSA and replace with fresh 1x PBS/BSA. 23. Repeat 15 min incubation at 4°C. 24. Spin down and resuspend Dynabeads in 200 ul lysis buffer (made fresh for the 2nd time that day because of decreased protease inhibitor half-life in aqueous solution). 25. Transfer 200ul 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 (cold room). Save remaining WCE at - 20°C for use in future steps.
Post-IP, elution, setup for reversal of crosslinks, annealing of linkers
1. Make fresh lysis buffer and 200 mM PMSF (150 mg + 4.3 ml 100% EtOH). 2. Make fresh high salt lysis buffer (360 mM NaCl final):
3.0 ml 0.5M HEPES-KOH pH7.5 2.16 ml 5 M NaCl 3.0 ml 10% Triton X-100 60 ul 0.5M EDTA 300 ul 10% Na-deoxycholate 30 ul PLAAC 150 ul 200 mM PMSF 21.3 ml ddH2O
3. Spin down Dynabeads/lysate at 3,000 rpm for 1 min at 4°C (cold room). 4. Use small magnet, SAVE supernatant and replace with 1 ml cold lysis (not high salt) buffer. SAVE the depleted supernatants for a Western to confirm that the IP worked. You will quantify the protein levels in the WCEs, their corresponding depleted supernatant, and will run it on a gel (along with some of the IP) to determine if your IP worked. 5. Incubate beads for 5 minutes (with lysis buffer) on rotator at 4°C. 6. Repeat the 1 ml lysis buffer wash--cold. 7. Wash 2x 1 ml cold "high salt” lysis buffer. 8. Wash 2x 1 ml cold "wash buffer":
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 9. To the rest of the samples, add 50 ul 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
10. 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. 11. Spin tubes at room temperature at 14,000 rpm for 30 seconds. 12. Using magnet to hold beads, transfer 30 ul of IP samples to a new tube--add 170 ul of TE/SDS.
1 ml Tris-HCl pH 8.0 10 mM Tris-HCl pH 8.0 200 ul 0.5 M EDTA 1 mM EDTA 1.6 ml 10% SDS 1 % SDS 98 ml ddH2O
Filter and store at RT
13. Since you do not use all of the IP, some will be available for checking by Western. 14. Thaw WCE from previous day and add 5 ul to a new tube--add 195 ul TE/SDS. 15. 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.
Prepare linkers - Not necessary to do this same day as the first DNA ppt will go several days, but is best done the day before you need them. 1. Need 6.7 ul mix per sample:
30 ul of 40 uM oJW102 (5'--GCG GTG ACC CGG GAG ATC TGA ATT C--3') 30 ul of 40 uM oJW103 (5'--GAA TTC AGA TC--3') 20 ul of 6x SSC (6x SSC on my bench as well as a bottle of 20x 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.
Post-decrosslinking, Proteinase K digestion, start of DNA ppt
1. Remove tubes from 65°C and prepare Proteinase K mix--let tubes return to room temperature before adding Proteinase K mix as you do not want to heat-denature your Proteinase K. 2. Proteinase K Mix (per sample)
single unit: 186.67 ul TE 4 ul glycogen (20 mg/ml, Roche) (freezer box) 10 ul Proteinase K (20 mg/ml, Gibco BRL) (freezer box)
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 ul of Proteinase K mix to each sample and incubate tubes for 2 hours at 37°C. 4. Transfer samples (~400 ul) to 2.0 ml Phase Lock gel tubes (Eppendorf) (on shelf near 50 ml conicals---tubes should be flash-spun before use just to get the gel down to the bottom of the tube). Add 400 ul of cold phenol:chloroform:isoamyl alcohol to each sample and pipette to mix. Do NOT vortex. 5. Spin at 13,000 rpm for 5 minutes at RT. 6. Transfer aqueous (~370 ul, everything above the gel layer which defines the boundary between aqueous and phenol) to a 1.5 ml Eppendorf tube. 7. Add 14.8 ul 5M NaCl to each (gives ~200 mM final concentration). 8. Add 2 volumes (740 ul) of cold 100% EtOH, vortex briefly, and incubate at -20°C for at least 2 days.
Post-DNA precipitation, RNase treatment, blunting, another precipitation
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 ul of TE (pH 8.0). To get the DNA fully into solution, I've found that incubating the tubes at 50°C (adjustable water bath at the end of my bench) 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 ul of 10mg/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 ul of EB Buffer (10mM Tris pH 8.0). Have ~48 ul left here. As in the past, I add the 50 ul back to the filter and spin an extra time in an effort to recover as much DNA as possible. 8. Setup blunting-- in separate PCR tubes, combine 2 ul of WCE + 38 ul ddH2O. For the IPs, add 40 ul of immunoprecipitated DNA (no H2O needed). 9. To each, add 70 ul of:
11 ul 10x T4 DNA pol buffer 0.5 ul 10mg/ml BSA 0.5 ul dNTP mix (20 mM each, made fresh immediately before use) 0.2 ul T4 DNA pol (3U/ul) 57.8 ul ddH2O
70 ul total
10. Mix by pipetting and incubate at 12°C for 20 min in a PCR block. DO NOT use the heated lid option. I have a program already called 12for20. 11. Place on ice and add 12 ul of
11.5 ul 3M NaOAc pH 5.2 0.5 ul glycogen (20mg/ml)
12 ul total
12. Mix by vortexing and transfer to a 0.5 ml Phase-Lock Gel extraction tube (above Seth's bench in Rm926). Add 120 ul of PCI and mix by pipetting. 13. Spin for 5 min at 13,000 rpm, room temp. 14. Transfer ~110 ul to a new 1.5 ml tube and add 230 ul ice cold 100% EtOH. 15. Incubate at -20°C for at least 2 h (The longer the better, shoot for two days). 16. Order Fresh Cy3-dUTP and Cy5-dUTP.
1. Spin for 30 min at 14,000 rpm at 4°C (large centrifuge in the cold room) 2. On ice, pipette off supernatant and wash the pellet with 500 ul cold 70% EtOH (in the door of my -20°C freezer). 3. Spin for 20 min at 14,000 rpm at 4°C (large centrifuge in the cold room) 4. On ice, pipette off supernatant, flash spin and remove any remaining liquid. Cover and allow to air dry at room temperature for 2 hours. 5. Resuspend pellet in 25 ul ddH2O (use the 60°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 ul of cold ligase mix to each tube (make a stock solution, 5x or 6x for 4 samples):
9.67 ul ddH2O 5 ul 10x DNA Ligase Buffer 6.7 ul Linkers (mix of 40 uM each, this is in my 4°C fridge) 0.5 ul T4 DNA Ligase 3.33 ul 15 mM ATP
Makes 25.2 ul total (add 25 ul to each tube)
7. Mix by pipetting and spin down to make sure all the liquid is at the bottom and incubate at 16°C in fridge near the Spiegelman lab. - Let this go over the weekend.
Post-ligation, PCR amplification, fragment size check and purification
1. Add 6 ul of 3M NaOAc (pH 5.2) and 130 ul 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 screw up the PCR later). 2. Pipette off supernatant and wash with 500 ul 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 (2h). 5. Resuspend pellets in 25 ul ddH2O and place on ice (60°C, vortexing as before), transfer to PCR tubes. 6. Add 25 ul of PCR mix to each tube:
5 ul 10x Thermopol buffer (NEB) 11.75 ul ddH2O 5 ul dNTP (2.5 mM each of all 4) 1.25 ul oligo oJW102 (40 uM stock) 1 ul Amplitaq polymerase (5 U/ul) Perkin-Elmer Cat. #N801-0060 1 ul Pfu Turbo dilution (dilute 1 ul of Pfu Turbo, 2.5 U/ul, in 20 ul ddH2O)
25 ul total
7. 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
8. After PCR amplification, check product on gel: 5 ul DNA, 5 ul H2O, 2 ul 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 minutes at 100V, look for a smear around 350-400bp. 9. Use Qiagen PCR purification kit and elute in 65 ul of their elution buffer.
BioPrime Incorporation of Dyes
Cy5-dUTP = IP = blue Cy3-dUTP = WCE = pink
1. Transfer 21 ul of DNA to an eppendorf tube. 2. Add 20 ul 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 ul of 10x dNTP (low T mix). BioPrime dNTP Mix (make your own-don't use theirs)
1.2 ul dATP 1.2 ul dCTP 1.2 ul dGTP 0.6 ul dTTP 95.8 ul ddH20
5. Add 3 ul of Cy5-dUTP (to IP sample), and 3 ul of Cy3-dUTP to WCE sample. (do in the dark---dyes are light sensitive) 6. Add 1 ul of high concentration Klenow (from BioPrime kit), make sure to mix well. 7. Incubate at 37°C for 1 h (wrap in aluminum foil-keep dark). 8. Use Qiagen purification kit to purify the DNA, elute in some considerable amount of EB buffer from kit (100 ul 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). 9. Add 12 ul of 3M NaOAc, 260 ul of 100% EtOH. Incubate at -20°C for at least a couple of days (wrap rack in aluminum foil). Pellet the DNA: 10. Spin at 14k rpm, 4°C, 40 min. 11. Wash with 500 ul 75% EtOH 12. Spin at 14k rpm, 4°C, 20 min. 13. Air Dry. Should have nice, tight, bright pellets. 14. Proceed to Microarray Hybridization