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Transient Hyper-Acetylation using Trichostatin-A treatment on S. cerevisiae cell cultures

Solutions :

SD+All (MCM)

Trichostatin A (Wako, 5 mg (204-11991)) :

Respuspend in Ethanol, insolvable in water. Keep at -20°C

Trichostatin A : 50X : 5mg in 11mL EtOH 50%

TBS1X : Make a dilution from the stock TBS10X (MCM). Autoclave

 

Day 0 :

Inoculate 50mL SD+All with one fresh single colony.

Let grow overnight at 30°C with shaking

Day 1 :

Take the OD (100µL of o/n culture in 900µL of water)

Adjust cultures at OD=0,15 (OD=0,3 Ultrospec) in 110mL SD+All

Let cultures grow at 30°C with shaking to have OD=0,5-0,6 (about 5h) (OD=1,08 on Ultrospec)

Take 45mL , centrifuge 5 min at 3500 rpm, remove all the supernatant and store the pellet at -80°C (to make protein extract later) Sample #1

Add Trichostatin A (TSA) to the remaining culture (65mL) to have 90µM final (3X1,3mL of TSA 50X) 

Let treat at 30°C with shaking during about 3-4 doubling time or about 8h (OD=3)

After TSA treatment : Take 20mL , centrifuge 5 min at 3500 rpm, remove all the supernatant and store the pellet at -80°C (to make protein extract later) Sample #2

Wash twice the remaining culture (about 45mL) with 20mL TBS1X

Resuspend in 1 mL TBS1X

Take 1/100 of the cells (10µL) to inoculate 50mL SD+All

Let grow overnight at 30°C with shaking.

Day 2:

At 11h30 : Take 10 µL from the overnight culture and inoculate a new starter culture in 50mL SD+All.

Take 20mL of the overnight culture , centrifuge 5 min at 3500 rpm, remove all the supernatant and store the pellet at -80°C (to make protein extract later) Sample #3

At 17h45 : Take the OD600nm, inoculate (vol=80/DO for BY and vol=20/DO for RM) in 200mL SD+All

Let grow overnight at 30°C with shaking.

Day 3:

When OD600=0,9 :

Take 25mL , centrifuge 5 min at 3500 rpm, remove all the supernatant and store the pellet at -80°C (to make protein extract later) Sample #4

With the remaining culture : Follow the « Single-Nucleosome Resolution Chromatin Immunoprecipitation from Yeast » protocol.

With the 4 samples : Follow the Western Blot protocol: control loading with anti-H3 or anti-GAPDH antibody, and visualize acetylation level with anti-H3K14ac antibody. Do NOT use anti-actin antibody for loading control, as TSA treated cells under-express actin!

Dernière mise à jour : ( 13-01-2010 )