ChIP in Yeast (for ~ 20 ChIPs)
1. Prepare chromatin
Grow a 2ml culture of the strain of interest ON
Inoculate a 20ml culture the next morning and grow ON
Dilute the culture 10 times and incubate until appearance of small buds (~2h)
Centrifuge the culture and wash with water
Resuspend in x ml of medium with appropriate treatment and incubate as long as needed (your cuisine)
Crosslink with 1% formaldehyde 30min at RT
Quench formaldehyde with 125mM glycine 5min at RT
Spin cells and resuspend in 20ml of ice cold TBS and repeat this washing step. From that step samples must stay ice-cold.
Centrifuge and resuspend cells in 500 to 1000µl lysis buffer (depends on the volume of the pellet)
Add this mixture to 500µl of acid washed glass beads and vortex on a bead mill 2 X 20min at 4C with 5min on ice in between
Collect samples by puncturing the lid and the bottom of the tube with a needle and centrifuge 1min at 1000rpm in a 15ml falcon tube
Shear chromatin by sonication: 2 X 15min at maximum output with 5min on ice in between using the Bioruptor Tween sonicator.
Centrifuge 10min at max speed
2. Prepare Magnetic beads
Mix 10µl of prot A and 10µl prot G precoated (20µl total) magnetic beads for each IP and wash in PBS+5mg/ml of BSA, repeat 1X
Incubate beads with antibody 1h at 4C
Wash 3X in PBS+5mg/ml of BSA and ressupend in lysis buffer
Distribute to as many law protein binding tubes as you have samples
3. Perform IPs, washes and elute
Add equal amounts of chromatin to the beads and add lysis buffer up to 800μl, incubate 2h to ON at 4C. If performing ON incubation, it is better to incubate ON with the ab only, and add beads the following day to collect the complexes 1h at 4C.
Wash 2X with lysis buffer (3 min on roller)
Wash 2X with wash buffer I (3 min on roller)
Wash 2X with wash buffer II (3 min on roller)
Wash 1X with TE (3 min on roller)
Centrifuge, resuspend in 50µl of elution buffer and incubate 10min at 65C with agitation, repeat 1X and pool elutions
Add RNase DNase free (Roche) up to 0,2µg/ml and incubate 8h to ON at 65C to reverse the crosslink
Add 3µl of proteinase K (20mg/ml SIGMA or Roche) and incubate 1h at 37C
Purifiy the DNA with Qiagen PCR purification kit and performe qReal Time PCR
Grow a 2ml culture of the strain of interest ON
Inoculate a 20ml culture the next morning and grow ON
Dilute the culture 10 times and incubate until appearance of small buds (~2h)
Centrifuge the culture and wash with water
Resuspend in x ml of medium with appropriate treatment and incubate as long as needed (your cuisine)
Crosslink with 1% formaldehyde 30min at RT
Quench formaldehyde with 125mM glycine 5min at RT
Spin cells and resuspend in 20ml of ice cold TBS and repeat this washing step. From that step samples must stay ice-cold.
Centrifuge and resuspend cells in 500 to 1000µl lysis buffer (depends on the volume of the pellet)
Add this mixture to 500µl of acid washed glass beads and vortex on a bead mill 2 X 20min at 4C with 5min on ice in between
Collect samples by puncturing the lid and the bottom of the tube with a needle and centrifuge 1min at 1000rpm in a 15ml falcon tube
Shear chromatin by sonication: 2 X 15min at maximum output with 5min on ice in between using the Bioruptor Tween sonicator.
Centrifuge 10min at max speed
2. Prepare Magnetic beads
Mix 10µl of prot A and 10µl prot G precoated (20µl total) magnetic beads for each IP and wash in PBS+5mg/ml of BSA, repeat 1X
Incubate beads with antibody 1h at 4C
Wash 3X in PBS+5mg/ml of BSA and ressupend in lysis buffer
Distribute to as many law protein binding tubes as you have samples
3. Perform IPs, washes and elute
Add equal amounts of chromatin to the beads and add lysis buffer up to 800μl, incubate 2h to ON at 4C. If performing ON incubation, it is better to incubate ON with the ab only, and add beads the following day to collect the complexes 1h at 4C.
Wash 2X with lysis buffer (3 min on roller)
Wash 2X with wash buffer I (3 min on roller)
Wash 2X with wash buffer II (3 min on roller)
Wash 1X with TE (3 min on roller)
Centrifuge, resuspend in 50µl of elution buffer and incubate 10min at 65C with agitation, repeat 1X and pool elutions
Add RNase DNase free (Roche) up to 0,2µg/ml and incubate 8h to ON at 65C to reverse the crosslink
Add 3µl of proteinase K (20mg/ml SIGMA or Roche) and incubate 1h at 37C
Purifiy the DNA with Qiagen PCR purification kit and performe qReal Time PCR