CHIP PROTOCOL FOR YEAST

CHROMATIN IMMUNOPRECIPITATION (CHIP) PROTOCOL FOR YEAST This protocol is derived from a paper by Miriam Braunstein and is based on work in the Allis lab. The procedure was written by Pam Meluh and updated by Paul Megee (6/10/00). References: •Dedon, P. C., J. A. Soults, C. D. Allis, and M. A. Gorovsky. 1991. A simplified formaldehyde fixation and immunoprecipitation technique for studying protein-DNA interactions. Anal. Biochem. 197: 83-90. •Braunstein, M., A. B. Rose, S. G. Holmes, C. D. Allis, and J. R. Broach. 1993. Transcriptional silencing in yeast is associated with reduced nucleosome acetylation. Genes & Devel. 7: 4, 592-604.

2 Days Before--Start 5 mL overnight from single colony. Day Before--Subculture overnight into 110 mL (large flask for aeration). Dilute so ~2x107 cells/mL at convenient time on Day 1, or if a cell cycle arrest will be performed, subculture into 220 mL and have cells at ~O.D. 0.5 at the time of addition of alpha factor, HU or NZ. Reserve Sorvall centrifuge (SS34, 4°C). Cool large rotor (TMA-3E) for TOMY centrifuge.

Day 1

When culture is ready (usually I aim for OD600 of 1.2; I’ve used up to 1.5), add formaldehyde directly to the medium--1% final concentration. Calculation: VOL of culture/36 = mL 37% H2 C=O. Fix at room temperature with gentle swirling (at least occasionally). **The time of fixation is an experimental variable. For Mif2p, yields of CEN DNA in the eventual anti-Mif2p IP increase with increasing fixation time. Earlier protocols used short fixation times (e.g. 10 minutes). For MCD1-6HA or MCD1-18MYC, 2 h. fixation worked well. Transfer cells to 50 mL or 250 mL conical tubes and spin down in clinical centrifuge. If cells will not be processed until the next day, wash twice with 25 ml cold PBS (transfer to a Nalgene Oak Ridge tube) and resuspend cells in 10 ml PBS and place at 4°C overnight. SPHEROPLAST CELLS. Volumes are for 100 mL of cells. (I have used the same volumes for 220 ml of staged cells successfully). Resuspend cells by gently vortexing (avoid pipetting). Resuspend cells in a total of 5 mL 0.1 M TRIS (pH 9.4), 10 mM DTT (freshly prepared from 1M DTT stock. Place on ice for 15-20 minutes. Spin down in Sorvall--5 K, 5 minutes, 4°C. Drain. Resuspend cells in 5 mL HEPES/sorb to wash. Spin down (5 K, 5 minutes, 4°C). Drain. Resuspend in 5 mL HEPES/sorb with 0.5 mM PMSF (add all inhibitors immediately before use). Add 60 µL 1 mg/mL oxalyticase (can also use 2 mg zymolyase here, as preferred). Incubate at 30°C for 20 minutes with gentle agitation. Check spheroplasting microscopically. Add additional oxalyticase if needed. When spheroplasting is complete, add 10 mL PIPES/sorb with 0.5 mM PMSF. If spheroplasting of some samples is completed earlier, add PIPES/sorb, invert and place on ice until all are complete. Spin down immediately--5 K, 5 minutes, 4°C. Drain. WASH SPHEROPLASTS (3x). All manipulations on ice. Use ONLY plastic pipettes to resuspend pellets. GENTLY resuspend cell pellet in 5 mL cold PBS with 0.5 mM PMSF. Spin down @ 5 K, 5 minutes, 4°C. Drain. Gently resuspend cell pellet in 5 mL cold Triton X/HEPES with 0.5 mM PMSF, 0.8 µg/mL pepstatin A, and 0.6 µg/mL leupeptin. Spin down @ 7 K, 7 minutes, 4°C. Drain. Gently resuspend cell pellet in 5 mL cold NaCl/HEPES with 0.5 mM PMSF, 0.8 µg/mL pepstatin A, and 0.6 µg/mL leupeptin. Spin down @ 7 K, 7 minutes, 4°C. Drain. SONICATE SPHEROPLASTS. Do not dawdle; resuspend quickly and transfer to 2 ml round bottom Eppendorf. Resuspend cell pellet in 1 mL SDS lysis buffer with 1 mM PMSF, 0.8 µg/mL pepstatin A, and 0.6 µg/mL leupeptin. Transfer to 2ml Eppendorf tubes. Sonicate suspension on ice for 10 second intervals, with at least 5 minutes on ice in between, until DNA is in 100-2000 bp range (average size should be 400-500 bp). For our Branson Sonifier 250: constant output @ 15-20% power, 6 pulses, 10 sec each. Spin in microfuge at maximum speed, 10 minutes, 4°C-10°C (TOMY @ 15K 18,000 x gmax). CHROMATIN SOLUTION Transfer sup (might seem cloudy from the SDS) to 15 mL Falcon snap-cap tube on ice (expect ~1.1 mL). (Tubes: VWR cat. # 60818-689) Add 10 mL IP Dilution Buffer with 1 mM PMSF, 0.8 µg/mL pepstatin A, and 0.6 µg/mL leupeptin (10 mL 9 volumes SDS lysate; final [SDS] 0.1%). Let sit on ice a while (approx. 20 min.). Spin in TOMY @ 10K (8,400 x gmax), 10 minutes, 4°C-10°C. Decant sup into 15 mL conical tube. Place on ice. This is the chromatin solution. Set aside 300 µl of chromatin as TOTAL. SET UP IMMUNOPRECIPITATIONS Aliquot chromatin solution to Eppendorf tube (I use 1.5 mL per IP; ~15-20 O.D.600 equivalents). Add appropriate volume of primary antibody. Incubate overnight 4°C on Nutator. I usually set up duplicate IPs. Note: Always do NO Ab control. Also, if adding competitor antigen, do so several minutes before adding Ab. It may be desirable to denature antigen in SDS first. In this case keep track of the additional SDS and supplement companion IPs to the same final [SDS]. Block beads with BSA. Remove 40 µl of a 50% slurry of swelled beads per immunoprecipitation to a 15 ml conical tube. Spin gently in a clinical centrifuge (~2K, 5 minutes), and remove supernatant with a P1000. Wash beads in 5 ml of bead blocking buffer (TE/0.1 % BSA/0.1% azide) and spin as before. Remove supernatant, add another 5 ml of buffer and place on rocking platform with immunoprecipitations.

Day 2 HARVEST IMMUNE-COMPLEXES

Add 4 µg lambda DNA (previously sonicated to 100-2000 bp size range). Use a razor blade to remove approximately 2 mm from the ends of pippette tips and use these to add 40 µL of Protein A Sepharose beads (prepared as 50% slurry in TE/0.1 % BSA/0.1% azide) to each tube. Incubate 1-2 hr. @ room temp. on Nutator. WASH IPs Spin down beads (2K, 2 min, 20°C). Remove aliquots of IP sups (0.5 mL), if desirable. Aspirate remaining sup. Wash beads sequentially with 1 mL of each of the following buffers, nutating beads 3-5 minutes in each buffer: TSE-150 TSE-150 or TSE-500 LiCl/Det TE TE** **Transfer beads to new Eppendorf with the second TE wash (0.5 mL for transfer + 0.5 mL to rinse old tube and tip). ELUTE IMMUNE-COMPLEXES After final IP wash, aspirate as much liquid as possible. Add 250 µL Bead Elution Buffer (1%SDS/0.1 M NaHCO3). Vortex briefly, then incubate 15 minutes @ RT on Nutator. Spin down beads. Carefully transfer sup to new Eppendorf, avoiding beads. Be patient here and wait for liquid clinging to pipet tip wall to drain. Add another 250 µL 1%SDS/0.1 M NaHCO3 to beads, repeat incubation, and combine sup with first elution. Use a fine pipet tip to aspirate liquid remaining in the beads and add to combined sups. Optional: After pooling eluates, spin to clear residual Protein A Seph., and transfer sup to new tube. REVERSE FORMALDEHYDE CROSSLINKS Add 5 M NaCl to samples: 20 µL (1/25 vol.) for eluted immune-complexes (i.e. Pellets); 2.5 µL for 0.3 mL aliquots of Total chromatin solution. Vortex and briefly spin. Incubate at 65°C for 4-5 hr. Add 2 volumes absolute EtOH. Precipitate overnight @ -20°C.

Day 3

Spin down EtOH ppts. Wash with 70% EtOH. Dry briefly in speed vac. Resuspend in 100 µL TE. Let sit on ice a while. Totals will be lumpy and gunky--break up with pipet tip, but do not pipette. Be careful not to lose material on the tip. Add 25 µL 5x Proteinase K Buffer, mix, then add 1.5 µL Proteinase K solution (Boehringer Mannheim). Incubate at 42°C for 1-2 hr. Aggregates in Totals should disappear. Add 175 µL TE to Pellet samples (final vol = 300 µL) and 275 µL TE to Totals (final vol = 400 µL) ORGANIC EXTRACTIONS Make fresh phenol:chloroform:isoamyl alcohol (25:24:1; PCI). Extract Pellets once with 300 µL PCI, then once with 300 µL CHCl3 . Extract Totals and Sups twice with 400 µL PCI and twice with CHCl3 . EtOH PRECIPITATION Add glycogen (2-5 µg) to Pellets. Add 1/10 volume 3 M NaOAc and 2 volumes absolute EtOH to all samples Precipitate overnight at -20°C.

Day 4

Spin down EtOH precipitates. Wash with 70% EtOH. Dry briefly in speed vac. Resuspend Pellets in 150 µL TE (equivalent to 10 µL chromatin solution per 1 µL). Resuspend Totals in TE volume equivalent to 1 µL chromatin solution per 1 µL (e.g. 300 µL TE for Total corresponding to 300 µL chromatin solution) Analyze samples for the presence of various DNA sequences by Slot Blot, Southern, or PCR. Dilute Totals 1:9 in TE before PCR analysis. Slot Blot Dilute aliquot of each sample in 6xSSC. Denature at 100°C ~10 minutes. Place immediately on ice. Apply samples to Nytran. Wash 2x with 6xSSC. UV crosslink filter prior to hybridization. Southern Blot Usually need to digest at least 1/2 of Pellet samples to see a signal; therefore, aliquot 75 µL to new Eppendorf tube and reduce volume in speed vac. Alternatively, if Southern blot analysis is anticipated, resuspend Pellets in a smaller volume initially. For Totals and Sups, digest an amount of chromatin solution equivalent to 1/5 to 1/10 amount used for Pellets. For CEN3, AluI is the diagnostic digest. Digest for several hours to overnight. Add RNase to bluejuice for Totals and Sups prior to loading onto gel. Run a 2.5% agarose gel. Transfer to GeneScreen. (I leave out the HCl treatment when preparing gels for transfer.). UV crosslink prior to hybridization. PCR I use 3 µL of each sample to program a 50 µL PCR reaction. (Note: Given the volumes used to resuspend various samples, the chromatin solution equivalent for Total is 1/10 that used for Pellet). Controls include no DNA and plasmid or good genomic DNA as a positive control.

If the experiment requires much PCR analysis, pellets can be diluted 1:4 in TE, with an equivalent dilution of totals (1:36). 3 µl of diluted template with 26 cycles of PCR will remain within a linear range for quantitation. Add 5x Bluejuice. Analyze 1/3 to 1/2 reaction on 2.5% agarose gel or 8% PAGE. Remember to be quantitative!! NuSieve GTG Agarose Gels PCR products are run on a 2.5% NuSieve GTG agarose gel (this works well for the typical size range of ChIP PCR products- 150-500 bp. For the large gel trays (20cm x 25cm), 200ml of 2.5% agarose is required. Weigh out agarose and place in a large flask to prevent boiling over during microwaving, and add 1X TBE buffer. The manufacturer recommends using cold buffer, but I have not found this to be critical. Stir the agarose solution for 20 minutes before microwaving. Weigh the flask before microwaving so that water lost during microwaving can be added back to the agarose before pouring. Use the following microwave settings: 3 minutes at power level 5, 30-45 at seconds highest power level. Stir the agarose to cool before pouring. Reweigh the flask and add water lost during heating. Just before pouring (I generally pour the gels in the cold), add ethidium bromide to a final concentration of 0.15 µg/ml (3µl of 10 mg/ml stock). After 30-45 minutes in the cold, place gel in the gel box and add 2 L of 1X TBE containing 0.15 µg/ml ethidium bromide. Let the gel sit for a while before removing combs. I typically run my gels at 120V for 90 minutes. Using these conditions, the current during the run generally begins at 70 mA and finishes at 80 to 90 mA. Avoid running gels at voltages that generate currents greater than 90 mA to prevent diffuse bands and heating of the agarose.

Reagents Needed for ChIP