CnR_panTCF_HEKdBcat_24hS33YBcat_rep1
Homo sapiens
Protocols: Cells were harvested by incubation in Trypsin EDTA 0.25%, and washed with PBS, and subjected to three washes with nuclear extraction buffer (HEPES-KOH pH-8.2 [20 mM], KCl [10 mM], Spermidine [0.5 mM], IGEPAL [0.05%], Glycerol [20%], 1xPMSF).  HEK293T cells WT and ΔCTNNB1 were cultured in DMEM (41966-029, Gibco – Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (12133C, Sigma-Aldrich) and 10 U/ml Penicillin-Streptomycin (15276355, Gibco - Thermo Fisher Scientific). Cells were passaged by incubation in Trypsin EDTA 0.25% (25-200-056, Thermo Fisher Scientific).  Cells were pre-treated with the PORCN inhibitor LGK974 (10 nM) for 24 hours and subsequently stimulated with the GSK3 inhibitor CHIR99021 (10 µM) for 30 minutes, 90 minutes, or 4 hours to activate Wnt/β-catenin signaling. Chromatin binding of β-catenin, pan-TCF/LEF (LEF1, TCF7, TCF7L1, and TCF7L2), CBP/p300, HDAC1, H3K27ac, and H3K4me1 was assessed across conditions. An IgG control was included for all experiments.  For perturbation experiments, cells were additionally treated with either the CBP/p300 inhibitor A-485 (5 µM) or the HDAC inhibitor sodium butyrate (1 mM). For the rescue experiment, LGK treated ΔCTNNB1 cells were transfected with pcDNA3-S33Yβ-catenin (Addgene#19286) or the empty vector control for 24 hours.  Resulting SAM files were converted to BAM files using SAMtools (v.1.11), followed by mate fixing. PCR duplicates were identified and removed using the markup function of SAMtool. To improve read quality, reads mapping to mitochondrial DNA and genomic blacklisted regions were removed using BEDTools (v.2.30.0) intersect. The filtered BAM files were subsequently sorted and indexed using SAMTools.  For each sample, 250,000 nuclei were bound to 10 µL magnetic ConA beads (Cell Signaling Technology #93569) for 15 minutes at 4 °C with rotation, using the manufacturer's activation buffer and instructions. Nuclei bound beads were washed with wash buffer (Hepes pH 7.5 [20 mM], NaCl [150 mM], Spermidine [0.5 mM], 1xPMSF) and incubated 5 minutes with wash buffer containing EDTA [0.2 mM], before they were incubated over-night in wash body containing EDTA [0.2 mM], BSA [0.01%], Digitonin [0.025%] and 1:100 antibody. For the panTCF condition, antibodies against LEF1, TCF1, TCF7L1 and TCF7L2 were mixed at equal ratios of 0.8 µL per antibody. For a list of all used antibodies, please refer to table 1. The next day, nuclei were washed five times and incubated 45 minutes at 4 °C in wash buffer containing digitonin [0.025%] and pAG-MNase [0.6 μg/ml]. After additional five washes, nuclei were incubated in 50 µL wash buffer containing CaCl2 [2 mM] for exactly 30 minutes at 4 °C for digestion. Digestion was terminated by adding 3 µL of a 250 mM EDTA/EGTA mix, and the initial fragment release was initiated by the addition of 2 µL NaCl [5 M] per 50 µL sample, followed by incubation at 37 °C for 30 min. This initial release was set aside, and nuclei were incubated in 50 µL urea stop buffer (NaCl [100 mM], EDTA [2 mM], EGTA [2 mM], IGEPAL [0.5%], Urea [8.8 M]) and incubated 30 minutes at RT for a secondary release. Both releases were combined and fragments were purified using phenol chloroform.  Library preparation was performed using the KAPA EvoPrep Kit (Roche, #10096039001) according to the manufacturer's instructions. For end repair and A-tailing, 14 µL of DNA was used in 0.4x reaction volumes. Adapter ligation was carried out in 0.4x reaction volumes using 0.15 µM KAPA Dual-Indexed adapters, followed by a post-ligation cleanup with Mag-Bind TotalPure NGS beads at a 1x ratio. Libraries were resuspended in 10 mM Tris-HCl (pH 8.0) and amplified using 0.5x reaction volumes. A post-amplification cleanup was performed with 1x beads. Libraries were size-selected by electrophoresis on a 2% E-Gel EX agarose gel (Invitrogen, #G402022) for 10 minutes using the E-Gel Power Snap Electrophoresis System. DNA fragments between 150 and 500 bp were excised and purified using the GeneJET Gel Extraction Kit (Thermo Scientific, #K0691). Libraries were quantified using the Qubit High Sensitivity DNA kit (Thermo Scientific, #Q32854), and pooled.
ENA first public
2026-06-27
INSDC center name
1 Department of Biomedical and Clinical Sciences, Division of Molecular Medicine and Virology, Faculty of Medicine and Health Sciences, Linköping University, Linköping, Sweden 2 Wallenberg Centre for Molecular Medicine, Linköping University, Linköping, Sweden 3 Science for Life Laboratory – SciLifeLab, Linköping University, Linköping, Sweden
INSDC status
public
Submitter Id
E-MTAB-17186:CnR_panTCF_HEKdBcat_24hS33YBcat_rep1
broker name
ArrayExpress
cell line
HEK293T
cell type
human embryonic kidney cell
collection date
not collected
common name
human
developmental stage
embryo stage
disease
none
fraction
NextSeq2000-P2
genotype
CTNNB1 knock-out
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
kidney
replicate
1
sample name
E-MTAB-17186:CnR_panTCF_HEKdBcat_24hS33YBcat_rep1
scientific_name
Homo sapiens
stimulus
pcDNA3-S33Yβ-catenin (Addgene#19286)