Tuesday, November 18, 2003
Description Yeast Chromatin Immunoprecipitation Procedure Day 1 1. Start 5 ml o/n cultures of strains to be tested Day 2 1. Inoculate 50 ml of the desired media with a volume of a saturated o/n culture and grow o/n with shaking at 200 rpm. The volume of cells inoculated should be such that the culture reaches the desired A600 the next morning. (For example, 130 microliters of a wild-type strain/100 ml YPD inoculated at 5:00 pm will be at an A600 of ~1.2 at 8:00 am next morning) Day 3 1. Check A600 until desired cell density is reached (usually ~ 1.5) 2. Once the desired cell density is reached remove the culture from the shaker 3. To the 50 ml culture add 1.39 ml formaldehyde (1% final), mix by swirling 4. Incubate at room temperature 10-20 min with occasional swirling (Times ranging from 1min to hours for crosslinking ChIP samples have been used in the literature. It is important to treat all cultures with formaldehyde for the same length of time.) 5. Add 2.75 ml 2.5 M glycine (125 mM final), mix by swirling 6. Incubate for 5 min at room temperature 7. Pellet cells in GSA at 5000 rpm for 10 min, decant supernatant 8. Wash cells with 20 ml cold TBS+125 mM glycine 9. Wash cells with 20 ml cold TBS, decant supernatant 10. Resuspend cells in the TBS that remains in the bottle after decanting (add 0.5mL TBS if necessary), and transfer the cells to a Marsh 2.0 ml tube 11. Pellet cells in a cold microcentrifuge for 10-30 sec, remove all supernatant 12. Freeze cell pellet on dry ice and store at 70 degrees C Days 3 and 4 are easily combined if the cells are ready early in the day. Day 4 1. Resuspend cell pellets in 400 ul ChIP lysis buffer+protease inhibitors 2. To each sample add an equal volume (about 0.5 ml) of glass beads (425-600 micron diameter) 3. Lock every Marsh tube to ensure the tubes dont open during lysis 4. Lyse cells in the Breeden Lab FastPrep vortex 3 x 40 sec, with 40 sec pauses between runs, at level 4.5 M/s (A2-124 cold room) 5. Remove locks and pierce tube bottoms with a hot 20 G 1.5 needle (yellow) 6. Put pierced tubes into 15 ml Sarstedt tubes and spin in a cold clinical centrifuge at top speed for 1 min (the extract will transfer to the 15 ml tube leaving the glass beads in the 2ml tube which can then be discarded) 7. Emulsify the insoluble pellets with their supernatants and transfer to 2.0 ml round bottom tubes (the chromatin is primarily in the insoluble fraction) 8. Sonicate extracts in Breeden Lab water bath sonicator at level 5, 100% duty, 4 x 30 sec with 30 sec pauses on ice between runs (sonicator output should be approximately 20-25 when sonicating samples) 9. Clarify extracts in cold room microcentrifuge for 10 min at top speed 10. Transfer supernatants to 1.5 ml tubes and centrifuge again in cold room for 5-10 min at top speed 11. Transfer supernatants to 1.5 ml tubes 12. Quantitate extracts by Bradford assay: 1 ul of a 1:6 dilution works well in the assay for samples taken from cultures between OD 1 and 2 13. Freeze extracts on dry ice, and store at 70 degrees C Day 5 1. Thaw extracts on ice and transfer a volume of extract (1mg total protein) to a new 1.5 ml tube for IP 2. Include necessary controls (i.e. no Ab control; untagged strain + Ab) 3. Bring all 1mg aliquots to 200 ul with ChIP lysis buffer+protease inhibitors, mix well (thorough mixing is very important) 4. Transfer 2 ul from each 200 ul IP sample to a new 1.5 ml tube with 150 ul ChIP elution buffer and set aside until the IPs are complete (these are the INPUT samples) 5. Add Ab to each IP sample except for the no-Ab control, nutate in cold room for 3 hrs 6. When there is 30 min left from step 5 prepare rProtein A (or G) Sepharose beads &Mac183; Spin down beads and remove supernatant &Mac183; Wash beads twice with 1ml ChIP lysis buffer+protease inhibitors per 250 ul bead volume &Mac183; Resuspend beads after washing in an equal volume of ChIP lysis buffer+protease inhibitors to make 50% slurry (prepare 25% more beads than you calculate to be necessary) 7. Add 60 ul of 50% bead slurry to each IP sample, nutate in cold room 1hr 8. Spin beads down in cold room microcentrifuge for 10-15 sec, remove the supernatant, and wash with the following solutions &Mac183; 2x 1ml ChIP lysis buffer+protease inhibitors &Mac183; 2x 1ml ChIP high salt lysis buffer+protease inhibitors &Mac183; 2x 1ml ChIP wash buffer &Mac183; 2x 1ml 1xTE (8.0) 9. After the last wash draw off as much liquid as possible from the beads using a finely pulled Pasteur pipette 10. Resuspend the beads in 85 ul ChIP elution buffer and incubate at 65 degrees C and 950 rpm in a Thermomixer for 10 min 11. Spin down beads in microcentrifuge and transfer 75 ul of the supernatant to a new 1.5ml tube 12. Add 75 ul to the beads, repeat steps 10 and 11, and combine the two 75 ul elutions (IP) 13. For each extract being tested you should now have an IP sample (150 ul) and an IN sample (150 ul) plus any controls 14. Incubate all IP and IN samples at 65 degrees C overnight to reverse crosslinks Day 6 1. Add 750 ul Qiagen buffer PB to each IP and IN sample 2. Purify using the Qiagen PCR purification kit, elute DNA in 50 ul buffer EB 3. Analyze the samples by quantitative PCR using an appropriate protocol Recipes ChIP Buffers 2.5 M glycine (filter sterilize, store at room temperature) 1x TBS + 125 mM glycine (store at 4 degrees C) 1x TBS (store at 4 degrees C) ChIP lysis buffer (filter sterilize, store at 4 degrees C) 50 mM HEPES pH 7.5 140 mM NaCl 1% TritonX-100 0.1% Sodium Deoxycholate 1mM EDTA ChIP lysis buffer (High Salt) (filter sterilize, store at 4 degrees C) 50 mM HEPES pH 7.5 500 mM NaCl 1% TritonX-100 0.1% Sodium Deoxycholate 1 mM EDTA ChIP wash buffer (filter sterilize, store at 4 degrees C) 10 mM Tris pH 8.0 250 mM LiCl 0.5% NP-40 0.5% Sodium Deoxycholate 1 mM EDTA 1x TE pH 8.0 (filter sterilize, store at 4 degrees C) ChIP elution buffer (store at room temperature) 50 mM Tris pH 8.0 1% SDS 10 mM EDTA Protease inhibitors PMSF, Benzamidine, Pepstatin, Leupeptin, and Chymostatin Phosphatase inhibitors (store at 20 degrees C) Solution A (100x) for 10 ml 100 mM sodium pyrophosphate 0.45g 100 mM sodium orthovanadate 0.18g Water to 10 ml Solution B (100x) for 10ml 100 mM beta-glycerophosphate 0.29g 100 mM EGTA 0.38g 1M NaF 0.42g Water to 10 ml Supplies Tips (责任编辑:泉水) |