12-hour RA-induction Nanog ChIP, mouse KH2 ES cells, replicate 1
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
No description
Antigen
NA
Cell type
Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA
Attributes by original data submitter
Sample
strain
KH2 ES Cells, Cat. No. MES4304
age
12 hrs post-induction
dev_stage
Embryonic
sex
male
tissue
ES Cells
biomaterial_provider
Open Biosystems, Huntsville, AL, USA
cell_line
KH2
cell_subtype
mHoxa1-3FMS-4C2
growth_protocol
KH2 ES cells with epitope tagged Hoxa1 (3XFLAG-MYC) 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 with differentiation media (DMEM + 10% Serum + NEAA+ 0.03 µM RA) RA supplemented 33nm for another 12 hours.
sample_type
cell culture
treatment
ChIP was done according to the Upstate protocol with certain modification [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]. 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 Anti Nanog antibody (D1G10), cell signaling technology (8785S).