description: Protocols: See nucleic acid extraction protocol β-catenin ChIP on mESC was performed using previous procedures and antibodies as described before for HEK293T (Doumpas et al., 2019). Briefly, mESC were treated with increased CHIR up to 10 μM for 24 h to stimulated Wnt/β-catenin signaling. Ca. 10 × 106 cells per samples were cross‐linked in 20 ml PBS for 40' with the addition of 1.5 mM ethylene glycol‐bis(succinimidyl succinate) (Thermo Scientific, Waltham, MA, USA), for protein-protein cross‐linking, and 1% formaldehyde for the last 20' 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' 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 μg of anti‐β‐catenin (SantaCruz sc‐7199 or Cell Signaling) and 50 μL 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 deoxycholate, 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‐cross‐linked 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. Libraries were prepared starting with 5 ng of ChIP-enriched DNA as starting material and processed with the Illumina TruSeq ChIP kit according to manufacturer specifications. Library molarity and quality was assessed with the Qubit and Tapestation using a DNA High sensitivity chip (Agilent Technologies).
attributes.tag: ENA first public
attributes.value: 2021-06-18
attributes.tag: ENA last update
attributes.value: 2021-06-18
attributes.tag: External Id
attributes.value: SAMEA9135474
attributes.tag: INSDC center alias
attributes.value: Wallenberg Center for Molecular Medicine (WCMM) Department of Biomedical and Clinical Sciences (BKV) Division of Molecular Medicine and Virology (MMV) Faculty of Medicine and Health Sciences, Linkoping University, Linkoping, Sweden
attributes.tag: INSDC center name
attributes.value: Wallenberg Center for Molecular Medicine (WCMM) Department of Biomedical and Clinical Sciences (BKV) Division of Molecular Medicine and Virology (MMV) Faculty of Medicine and Health Sciences, Linkoping University, Linkoping, Sweden
attributes.tag: INSDC first public
attributes.value: 2021-06-18T00:14:12Z
attributes.tag: INSDC last update
attributes.value: 2021-06-18T00:14:12Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-10575:Bcat_TCFqKO_3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: mouse embryonic stem cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: TCF/LEF quadruple knock-out
attributes.tag: sample name
attributes.value: E-MTAB-10575:Bcat_TCFqKO_3