Chromatin Immunoprecipitation (ChIP)

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1 de Lange Lab Chromatin Immunoprecipitation (ChIP) Required Solutions IP Wash A 0.1% SDS 1% Triton X mm EDTA ph mm Tris-HCl ph mm NaCl 1 mm PMSF 1 µg/ml Leupeptin 1 µg/ml Aprotinin 1 µg/ml Pepstatin Add protease inhibitors right before use. IP Wash B 0.1% SDS 1% Triton X mm EDTA ph mm Tris-HCl ph mm NaCl IP Wash C 0.25 M LiCl 1% NP-40 1% Na-Deoxycholate 1 mm EDTA ph mm Tris-HCl ph x PBS 82 g NaCl 2 g KCl 17.4 g Na 2 HPO 4.7H 2 O 2 g KH 2 PO 4 H 2 O to 1 l ph should be 7.4 after 10x dilution. Lysis buffer 1% SDS 10 mm EDTA ph mm Tris-HCl ph mm PMSF 1 µg/ml Leupeptin 1 µg/ml Aprotinin 1 µg/ml Pepstatin Add protease inhibitors right before use. IP dilution buffer 0.01% SDS 1.1% Triton X mm EDTA 16.7 mm Tris-HCl ph mm NaCl 1 mm PMSF 1 µg/ml Leupeptin 1 µg/ml Aprotinin 1 µg/ml Pepstatin Add protease inhibitors right before use. 20x SSC per liter: g NaCl 88.2 g Na citrate ph to 7.0 with a few drops of 10 N NaOH. TE (4 th IP wash) 10 mm Tris-HCl ph mm EDTA ph 8.0 Glycogen From Roche # mg/ml SDS-Na Carbonate 1% SDS 0.1 M NaHCO 3 Make fresh from 10% SDS and 1 M NaHCO 3 Church Mix 500 ml 1 M NaPi ph 7.2 (see below) 2 ml 0.5 M EDTA ph g SDS 10 g BSA ddh 2 O to 1 l Heat to 55ºC to dissolve. Filter through a 0.45 µm filter. 1

2 de Lange Lab 1 M NaPi ph 7.2 In 2.3 liters (orange capped roller bottle filled to shoulder). 308 g Na 2 HPO 4 *7H 2 O 9.2 ml H 3 PO 4 add ddh 2 O to close to final volume and adjust ph to 7.2 with H 3 PO 4 adjust volume to 2.3 l Beads Protein G-Sepharose Amersham/Pharmacia # ), use 30 µl per IP, blocked with: 5 µg E.coli DNA (sheared) 30 µg BSA, per 30 µl beads In an Eppendorf tube, pipette enough slurry for 1.5x the number of samples you have. Add 3 µl of BSA (10 mg/ml) x 1.5x # samples and 0.5 µl of E.coli DNA (10 mg/ml) x 1.5 # samples. Incubate on wheel overnight with the IPs and use directly the next day. The remainder can be kept at 4ºC and reused, but add sodium azide before storage. ChIP Protocol Timeline Day 1: Prepare the lysate and start the o/n IPs. Day 2: Wash the IPs, reverse the crosslinks (4 hrs), and extract the DNA, ppt o/n.* Day 3: Resuspend the DNA, dot blot, and start the hybridization.* Day 4: Wash the membrane and expose (a few hours). *Days 2 and 3 can be combined. 2

3 Preparing the lysate 1. Grow cells to subconfluence. Set up experiment for 10 IPs. For primary cells, use at least ten 15 cm dishes. For Hela cells use about five 15 cm dishes. (For 10 IPs you typically want a 3-5 ml cell suspension with 2 x10 7 cells/ml; see step 9 below for cell numbers). 2. Remove media. Add 15 ml PBS/1% Formaldehyde (from 37% solution). 3. Swirl on platform min. 4. Add 2 ml 1.5 M Glycine (final concentration 0.2 M) to stop crosslinking. 5. Swirl at RT for 5 min. 6. Wash with cold PBS x Scrape cells in ~ 10 ml PBS into 50 ml conical tube. Spin down cells. 8. Combine cell pellets into 20 ml cold PBS with PMSF. Spin again. Place cell pellet on ice. (Cells can be counted at this step, but it is an underestimate because it s hard to separate the cells completely.) 9. Resuspend pellets in lysis buffer + protease inhibitors in a 15 ml conical tube to a concentration of 2x10 7 cells/ml, usually 3-5 ml (for Hela or HTC75 cells, 10 7 cells/ml is fine). The buffer needs to be at RT, but keep the lysate on ice from this point on. The lysate can be more concentrated if necessary, but no more than 5x10 7 cells/ml. For IMR90 cells, make a lysate at 5x10 7 cells/ml. 10. Incubate for 15 min on ice. 11. Sonicate on ice,10 cycles of 20 seconds each, 50% duty (0.5 s ON/OFF) at power setting 5, with the cell suspension on ice at all times. The lysate should clear up. Check between cycles to make sure that the lysate remains ice-cold. Take a 50 µl aliquot after the second cycle and after last cycle to check for sonication on a gel. To do this, reverse crosslinks and extract the DNA as outlined below. Bulk DNA should be sheared to kb. 12. After sonication, spin in clinical centrifuge at 3000 rpm for 5 min. Divide the supernatant in 1.5 ml aliquots in Eppendorf tubes and microfuge at max for 10 min. Pool superatants in a 15 ml conical. This is your lysate. 3

4 Immunoprecipitations 1. Use 200 µl lysate per IP. On ice, add 1.2 ml of IP dilution buffer + protease inhibitors (fresh). Mix by gently inverting the tubes a few times, let sit on ice for 10 min, and add the antibody. For our rabbit polyclonal sera, use crude serum and include the pre-immune controls. Usually µl of serum is good. 5 µl of 9E10 (Myc) or 2 µl of M2 (FLAG) gives good results for tagged TRF1 or TRF2. Incubate on wheel overnight at 4ºC. 2. At the same time, prepare enough beads for the next day. Per IP, use 30 µl of Protein G agarose beads, with 5 µg sonicated E. coli DNA and 30 µg BSA (refer to Preparation of E.coli DNA on page 6). Prepare enough beads for at least 10 samples extra as the mixture can be thick and hard to pipette. Pipette the beads using a tip with its end cut off (razorblade). 3. Remember: Keep two aliquots of 50 µl lysate (o/n at 4ºC), as total fractions, to be processed the next day. They will be processed with the other samples after the immunoprecipitation step and will be used as 100% values in the quantitation. 4. The next day: Add 30 µl prepared beads to each IP. Incubate another 30 min at 4ºC. 5. Wash the beads once with 1 ml of each of the buffers: A, B, C, and TE. Add protease inhibitors only to buffer A. Vortex and place on ice in between washes. Use table top centrifuge to spin down. 6. Elute the immunoprecipitated material as follows: At the end of the last wash, repellet the beads, remove as much buffer as possible, and resuspend beads with 250 µl of 1% SDS/0.1 M NaHCO 3 (freshly diluted) by vortexing. Incubate for 10 min at RT. Pellet the beads, transfer supernatant to a fresh tube, and wash the beads again with 250 µl of SDS-carbonate. Incubate at RT 10 for min, pellet beads, and pool supernatant with the previous eluate. 7. To the 500 µl eluate, add 20 µl of 5 M NaCl, mix, and incubate at 65ºC for 4 to 6 hours to reverse the crosslinks. Important: this is the step where the total fractions start to get processed. 8. For the total input samples bring the volume up to 500 µl with SDS-carbonate (i.e. add 450 µl to the 50 µl of lysate set aside), add 20 µl of 5 M NaCl and incubate at 65ºC with other fractions. 9. After 4-6 hours at 65ºC, add 10 µl of 0.5 M EDTA and 20 µl 1M Tris-HCl ph Add 20 µg RNAse A (20 µl of 1 mg/ml, heat inactivated, DNAse-free), incubate at 37ºC for 30 min. 11. Add 40 µg of Proteinase K (20 µl of 2 mg/ml freshly made stock). Incubate for 1 hour at 37ºC. 12. Extract the samples with 0.5 ml phenol/chloroform/isoamylalcohol. Transfer the aqueous phase to an Eppendorf tube and add 20 µg of glycogen (from 20 mg/ml stock) and mix. Add 1 ml of EtOH to precipitate the DNA. Precipitate o/n at -20ºC. 13. Spin down for 10 min in microfuge at max speed at 4ºC. Remove all EtOH. Resuspend pellet in 100 µl of ddh 2 O or 10 mm Tris-HCl ph

5 Dot Blotting and hybridization 1. For dot blotting, first prepare the minifold and membranes. Then boil DNA samples for 5 min, snap cool on ice, quick spin in microfuge, and load. 2. With the dot blot manifold, use two 3MM Whatman papers and Hybond-N membrane, all saturated with 2x SSC (wet Hybond first by floating on top of H 2 O, then soak in 2x SSC). Ensure the vacuum is working well by loading 2x SSC on a few wells. Then switch the vacuum off until you are ready to load. First pipet 200 µl of 2x SSC in each well. Then load the samples and mix with the 2x SSC. This ensures an even distribution of the sample on the membrane. Then switch on the vacuum. 3. Load 30 µl (80 µl for IMR90 cells) on dot blot to probe for telomeric DNA with Sty11 insert, load 10 µl on membrane to probe for Alu (human cells) or Bam repeats (mouse cells). 4. Load 1, 2, and 4 pg Sty11 plasmid to verify that the range is linear in the quantitation. The total fractions usually fall in that range. It is a good idea to load a serial dilution of the total fractions. 5. Denature the membrane by placing it on a 3MM Whatman saturated with 1.5 M NaCl/ 0.5 N NaOH. 6. Denature for 10 min. 7. Neutralize the membrane by placing it on a 3MM WHatman saturated with 1 M NaCl/ 0.5 M Tris-HCl ph 7.0 for 10 min. 8. Remove excess liquid by gently blotting on dry paper. Crosslink the DNA to the membrane immediately in the Stratagene UV crosslinker, DNA side up. Rinse in 2x SSC. 9. Prehybridize in medium size Hybaid bottle with 7 ml Church buffer for 30 min at 65ºC. 10. Add filtered mixture of Church mix and denatured probe (7 ml -- see Telomere Blot protocol) and hybridize o/n at 65ºC. A quarter of the probe prepared as usual is enough. 11. Washes: first wash the membrane in 2x SSC at RT for 5 min. Wash 2-3 times. If necessary, perform the following washes, but carefully monitor the blot after each wash. Expose as soon as non-specific counts appear to have disappeared (e.g. in the corners): 2x SSC/RT, 0.2x SSC/RT (usually this is enough), 2x SSC 0.1%SDS/RT, 0.2x SSC 0.1%SDS/RT. Perform same sequence of washes at 65ºC if necessary. 12. Expose on the phosphoimager (usually a few hours). For the quantitation, multiply the counts for the total fractions by 4 and use this value as 100%. 5

6 Note on detecting DNA by PCR PCR can be performed on 10 µl of the eluate after reversing the crosslink (no need for proteinase K and CPI). The PCR can also be done on the material after EtOH precipitation (use 5 µl). Preparation of E.coli DNA For use in EMSA experiments 1. Dissolve 25 mg E.coli DNA (Sigma D-2001, type VIII) in 10 mm Tris 7.5 at a final concentration of 2.5 mg/ml. 2. Sonicate (or shear 11 times with tuberculin syringe) to reach a ~400 bp fragment pool. The efficiency varies per batch, but it is best to do less so you do not overfragment. You can check ~1 µl on a gel after each sonication, and repeat until the desired size range is achieved. 3. CIAA extract 1 x. 4. CHCl3 extract 1 x. 5. Add 1/10 volume 1 M NaOAc, mix, and EtOH precipitate -- Wash well with 70% EtOH. 6. Resuspend with TE to ~3 mg/ml (estimated). 7. Take OD 260 to accurately quantitate. 8. Store at -70ºC. 9. Make 1 mg/ml working stock and store at -20ºC. 6