Illumina HiSeq 4000 sequencing; ChIP-seq experiment of mouse embryonic stem cells (mESCs), assessing beta-catenin DNA binding in wild type mESCs and TCF/LEF quadruple knock-out mESCs.
ChIP-seq experiment of mouse embryonic stem cells (mESCs), assessing beta-catenin DNA binding in wild type mESCs and TCF/LEF quadruple knock-out mESCs.
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).