Milea Timbergen

96 data repository under accession number PRJNA604749. The DMRs with a fold change ≥1.5 were considered to be differentially methylated and were analysed separately. Non-normal distributed values were analysed using a Mann-Whitney U test to identify statistically significant differences in the normalized read counts between the two mutation types. A p-value of < 0.05 was considered to be statistically significant. SPSS Statistics (version 24) was used for the Mann-Whitney U tests (IBM, Armonk, New York, USA). The DMRs of interest were loaded in the Integrative Genomics Viewer (IGV) using the Hg 38 platform to visualize regions of interest 30 . Validation MeD-seq results using an mRNA expression dataset Expression data, generated on an Affymetrix platform (Human Genome U133 Plus 2.0 array) of PERM1, DUX4L6, CCDC6, NOC4L, FOXK2 and NLRP4 in DTF samples (n = 128) were obtained from a publicly available dataset in the Gene Expression Omnibus (accession number GSE58697) 31 Information regarding the CTNNB1 mutational status was kindly provided by dr. Frederic Chibon, Cancer Research Center of Toulouse, France. Only patients with an S45F or T41A mutation were selected for validation purposes. A Mann- Whitney U test was performed on non-normal distributed data to identify differences in mRNA expression levels of the selected genes corresponding to the identified DMRs. A p-value of < 0.05 was considered statistically significant. SPSS Statistics (version 24) was used for all statistical analyses. Cell lines, cell transfection The human cell lines HEK293T (embryonic kidney cells) and HCT116 (colon cancer cells) were maintained in DMEM (Gibco Life Technologies) supplemented with 10% fetal bovine serum (Greiner bio-one) and 100 IU/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. For transfections cells were cultured in 6-well plates to 70% confluence. Next the cells in each well were transfected with 1 µg of different N-terminal FLAG tagged β-catenin plasmids or empty pcDNA 5’UT-FLAG vector using ViaFect™ (Promega) as transfection agent. The construction of the different expression plasmids is described by Liu et al. 32 . The CTNNB1 plasmid variants used express FLAG tagged versions of either the wild-type (WT), T41A, S45P, exon 3 deletion or K335I β-catenin. Cell lysates, Immunoprecipitation and Western blotting At 48 h post-transfection cells were washed with ice-cold PBS and lysed in 500 µl of lysis buffer 25 mM Tris-HCl pH 7.5; 150 mM NaCl; 1 mM EDTA; 1% NP-40 and 5% 4