KH2 ES cells were grown in feeder free condition using N2B27+2i media supplemented with 2000 U / ml of ESGRO (Millipore). N2B27+2i media consist of neurobasal media (21103-049, Invitrogen) , DMEM/F12 media (10565-018, Invitrogen), 0.5X N2 (17502-048, Invitrogen), 1X B27 (17504044, Invitrogen),1X b-mercaptoethanol (ES-007-E , Millipore), 1X Glutamax (10378-016) ,1X NEAA (07600, SCT), 3uM CHIR99021 (4423, Tocris), PD0325901 (72184, SCT), 0.033% BSA (15260037) Thermo Fisher scientific) (Qi-Long, 2008). Cells were seeded on gelatinized plate without feeder layer. After 48 hours media was changed to differentiation media (DMEM + 10% Serum + NEAA+ 0.03 µM RA) RA supplemented 33nm for another 24 hours.
sample_type
cell culture
label
24-hour RA-induction TGIF IP, replicate 1
treatment
ChIP was done according to the Upstate protocol described in [Smith KT, Martin-Brown SA, Florens L, Washburn MP, Workman JL. 2010. Deacetylase inhibitors dissociate the histone-targeting ING2 subunit from the Sin3 complex. Chem Biol 17: 65–74] with certain modifications. Cells were fixed by adding formaldehyde to media at a final concentration of 1% and by incubating at 37oC for 11 min. Crosslinking was stopped by 1mL 1.25 M glycine per 10 mL and by incubating at room temperature for 5 min. Cells were sonicated for 25 min in Bioruptor at high setting and 30 sec on-off cycle. ChIP was done using TGIF antibody, Abcam (ab170802).