Yeast Chromatin Immunoprecipitation (ChIP)
Hahn Lab
July 2003
Chromatin IP Method
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 400μl 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 don’t 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: 1μl of a 1:6 dilution works well in the assay for samples taken from cultures between OD1 and 2
13.Freeze extracts on dry ice,and store at -70 degrees C