Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
CTNNB1

Cell type

Cell type Class
Kidney
Cell type
293
Primary Tissue
Kidney
Tissue Diagnosis
Normal

Attributes by original data submitter

Sample

Alias
E-MTAB-7028:Exp1_WT1-Bcat
Broker name
ArrayExpress
Description
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 checklist
ERC000011
INSDC center alias
UZH
INSDC center name
University of Zurich
INSDC first public
2018-11-23T17:04:23Z
INSDC last update
2018-07-20T09:50:02Z
INSDC status
public
SRA accession
ERS2619215
Sample Name
ERS2619215
Title
Exp1_WT1-Bcat
cell_line
HEK293T
cell_type
epithelial cell
developmental stage
embryo
disease
normal
genotype
wild type genotype
organism
Homo sapiens
organism part
kidney
stimulus
GSK3 inhibitor CHIR99021 (10 micromolar) for 24 hours

Sequenced DNA Library

library_name
Exp1_WT1-Bcat_s
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
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.

Sequencing Platform

instrument_model
Illumina HiSeq 4000

hg19

Number of total reads
28090002
Reads aligned (%)
13.2
Duplicates removed (%)
85.9
Number of peaks
387 (qval < 1E-05)

hg38

Number of total reads
28090002
Reads aligned (%)
14.0
Duplicates removed (%)
85.1
Number of peaks
387 (qval < 1E-05)

Base call quality data from DBCLS SRA