Homo sapiens
Protocols: Human embryonic kidney (HEK) 293T cells were cultured in media consisting of DMEM, high glucose (41966-029 Gibco) supplemented with 10% fetal bovine serum (Gibco), 1x penicillin‐streptomycin according to the manufacturer's recommendation. We performed ChIP using an anti-beta-catenin antibody, followed by whole-genome sequencing (ChIP-seq), on both CHIR-stimulated WT and d4TCF cells. Cells were treated with 10μM CHIR or 10 μM DMSO for 24 hours prior to cross-link. Ca. 50 x 10^6 HEK cells per samples were cross-linked in 20 ml PBS for 40 min with the addition of 1.5 mM ethylene glycol-bis(succinimidyl succinate) (Thermo Scientific, Wal- tham, MA, USA), for protein-protein cross-linking (Schuijers et al., 2014.), and 1% formaldehyde for the last 20 min of incubation, to preserve DNA-protein interactions. The reaction was blocked with glycine and the cells were subsequently lysed 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). Chromatin was sheared using Covaris S2 (Covaris, Woburn, MA, USA) for 8 min with the following set up: duty cycle: max, intensity: max, cycles/burst: max, mode: Power Tracking. The sonicated chromatin was diluted to 0.15% SDS and incubated overnight at 4°C with 10 g of anti β-catenin (Santacruz sc-7199; Cell Signalling) and 50 l of protein A/G magnetic beads (Upstate). The beads were 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 deoxycho- late, 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), and finally twice with Tris EDTA buffer. The chromatin was eluted with 1% SDS, 0.1 M NaHCO3, de-crosslinked by incubation at 65°C for 5 h with 200 mM NaCl, extracted with phenol-chloroform, and ethanol precipitated. The immunoprecipitated DNA was used as input material for DNA deep sequencing.
ENA-FIRST-PUBLIC
2018-11-23T17:04:23Z
ENA-LAST-UPDATE
2018-07-20T09:50:02Z
External Id
SAMEA4799300
INSDC center name
UZH
INSDC first public
2018-11-23T17:04:23Z
INSDC last update
2018-07-20T09:50:02Z
INSDC status
public
Submitter Id
E-MTAB-7028:Exp2_dbcat-bcat
broker name
ArrayExpress
cell line
HEK293T
cell type
epithelial cell
common name
human
developmental stage
embryo
disease
normal
genotype
beta-catenin knockout
organism part
kidney
sample name
E-MTAB-7028:Exp2_dbcat-bcat
scientific_name
Homo sapiens
stimulus
GSK3 inhibitor CHIR99021 (10 micromolar) for 24 hours