Chromatin immunoprecipitation_by_Claudio Cantù

ChIP_Cantù

- Embryo tissue/cells are dissected and dissociated to a single cell suspension

- Cells are cross-linked in 20 ml PBS (RT) for 40 min with the addition of 1.5 mM ethylene glycol-bis(succinimidyl succinate) (Thermo Scientific, Waltham, MA, USA) (0.022 g for 20ml of solution - EGS is soluble in DMSO, and you will need 3ml of DMSO to dissolve 0.022 g - keep all solutions at room temp)

- 1% formaldehyde (MetOH-free) is added for the last 20 min of incubation

(For classical TF EGS could be avoided - go directly to formaldehyde and keep everything ice cold/4°C)

- The reaction is blocked with glycine (125 mM final concentration) - spin at 1200 rpm for 7’ - wash with PBS (not cold if there is EGS in the solution) and spin 1500rpm for 5’

- Cell pellet is resuspended in 1 ml hepes buffer (0.3% SDS, 1% Triton-X 100, 0.15 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES) + protease inhibitors

- Chromatin is sheared using Covaris S2 (Covaris, Woburn, MA, USA) for 8 min with the following set up: duty cycle: 20%; intensity: 10 (max); cycles/burst: 1000; mode: Power Tracking. 

- The sonicated chromatin is diluted to 0.15% SDS (add 1ml of Hepes buffer without SDS) and spun down 5’ was speed to remove cell debris.

- The supernatant is incubated overnight at 4°C with antibodies (1microg for chromatin marks, 10micrograms for TF) and magnetic beads (30-50 microL). 

- After ON incubation beads are washed at 4°C with: 

wash buffer 1 (0.1% SDS, 0.1% deoxycholate, 1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES)

wash buffer 2 (0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 0.5 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES)

wash buffer 3 (0.25 M LiCl, 0.5% sodium deoxycholate, 0.5% NP-40, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES)

twice with Tris EDTA buffer. 

- The chromatin is eluted and decrosslinked with 1% SDS, 0.1 M NaHCO3 + 200mM NaCl by incubation at 65°C for 4h/ON

- proteinase K (add pK, temp not higher than 60 degrees - addition of Tris EDTA is good (EDTA up to 25mM))

- DNA is extracted with phenol-chloroform, and ethanol precipitated. The immunoprecipitated DNA iss used as input material for DNA library prep and deep sequencing.