biostudies-arrayexpressMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-13574The experiment was designed to assess the DNA methylation differences between CLDN6Low vs CLDN6High EpiSCs and APSD, as well as WT vs Pbx1-KO EpiSCs. Based on analysis of RNAseq data and GO, we narrowed down our focus to DNA methylation effects influencing lineage biases. We performed WGBS analysis using two different methodologies. The EZ DNA Methylation-Lightning Kit (Zymo Research, #D5031) for bisulfite conversion of CLDN6Low versus CLDN6High sorted EpiSCs and APSD cells. The next steps of library preparation was done using NEBNext Ultra II DNA Library Prep Kit (E7645S), enzymatic fragmentation using NEBNext dsDNA Fragmentase (M0348S), and sensitive adaptors NEBNext Multiplex Oligos (Methylated Adaptor, E7535S), as per manufacturer’s instructions. Samples were sequenced on NextSeq2000 (200 cycles kit), averaging 256M reads per sample. WT versus Pbx1-KO EpiSCs were treated via the post-bisulfite library construction method using Illumina’s TruSeq DNA Methylation Kit (EGMK81312) with sensitive adaptors (EGIDX81312) following the manufacturer’s instructions.biostudies-arrayexpressSequencing - Samples were sequenced on NextSeq2000 (200 cycles kit), averaging 256M reads per sample.Sample Collection - EpiSCs were seeded at ~6 x 103 cells/cm2 in 6-well cell-culture plates pre-coated with 15 µg/ml human Fibronectin. Cells were dissociated using Accutase for 3 min at 37°C and counted. 150 x 106 cells per condition were incubated with the CLDN6 Alexa Fluor 488-conjugated antibody (R&D Systems, FAB3656G, 1:200) and Ghost Dye™ Red 780 (Tonbo, 13-0865, 1:1000) for 20 min on ice, washed with ice-cold FACS buffer (10% (v/v) FBS in PBS) and resuspended into 1 ml. Cells were sorted into MACSQuant Tyto Running Buffer using the MACSQuant Tyto sorter (Militenyi Biotec) or into ice-cold FACS buffer using the SONY SH800S cell sorter (Sony Biotechnology) at the DanStem/reNEW Flow Cytometry Platform (University of Copenhagen, Copenhagen, Denmark). All reseeding experiments were performed using the MACSQuant Tyto (Militenyi Biotec) sorter, taking advantage of high-speed, multi-parameter flow sorting in the safety of a fully enclosed cartridge to prevent contamination.Sample Treatment - All differentiation was conducted in serum-free, feeder-free and monolayer conditions in chemically-defined N2B27-based media. EpiSCs were seeded at ~10.5 x 103 cells/cm2 in 6-well cell-culture plates pre-coated with 15 µg/ml human Fibronectin, and cultured in EpiSC medium for 24h. EpiSCs were differentiated towards either APSD using 30 ng/ml Activin A, 4 µM CHIR-99021, 20 ng/ml bFGF and 100 nM PIK-90 (APS medium) for 24 For NMP/PSM differentiation, EpiSCs were first differentiated towards APSD, and then to NMP/PSM using 1 µM A-83-01, 3 µM CHIR-99021, 250 nM LDN193189 and 20 ng/ml bFGF for 48-72h (PSM media). For DE differentiation, EpiSCs were first differentiated towards APSD in N2B27-basal media over 24 h, and then to DE using 30 ng/ml Activin A, 4 µM CHIR-99021 and 100 nM PIK-90 in RPMI(Glutamax) + B27 (Without insulin) basal media (DE medium) for 48 h. EpiSCs were differentiated towards neuroectoderm based on known media requirements (PMID: 20207225). Differentiation of EpiSCs towards PPS and ExM were done as previously described (PMID: 36142249)Nucleic Acid Extraction - DNA was extracted from sample pellets using the Qiagen DNeasy Blood & Tissue Kit following the manufacturer’s instructions. 1 μg of sample gDNA was used to prepare the libraries.Library Construction - We utilized the EZ DNA Methylation-Lightning Kit (Zymo Research, #D5031) for bisulfite conversion. CLDN6Low versus CLDN6High WGBS library preparation was done using NEBNext Ultra II DNA Library Prep Kit (E7645S), enzymatic fragmentation using NEBNext dsDNA Fragmentase (M0348S), and sensitive adaptors NEBNext Multiplex Oligos (Methylated Adaptor, E7535S), manufacturer’s instructions.Growth Protocol - Post-sorting, cells were reseeded into Fibronectin pre-coated 6-well cell culture plates at around 10.5 x 103 cells/cm2.MINSEQE ScoreAssays and DataProcessed DataorganisationMAGE-TAB FilesSequence Alignment - Adapter trimming, sequence alignment, and methylation calls were performed using Bismark (v0.18.1).Data Transformation - Differential methylation analyses were performed using methylKit (v1.25.0), applying Fisher's exact test for comparing the fraction of methylated Cs in differentially probed samples, with cutoff for the absolute value of methylation percentage change (25%) and qvalue of differential methylation statistic (0.01). Differentially methylated Cs were annotated with rGREAT (v4.0.4) using single nearest gene approach and defined as differentially methylated regions near genes (DMGs). The coverage cutoff was set at 10, and 4 For CLDN6 sorted WGBS data and WT versus Pbx1 WGBS data, respectively. The output from Bismark was also imported into SeqMonk (v1.46.0; Babraham Bioinformatics) for parallel DSS-based methylation validation analyses and WGBS data visualization over key genes of interest.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsNextSeq 2000MacLab Benchmethylation profiling by high throughput sequencingMus musculusDifferential ERK activity shapes the epiblast methylome influencing cell-context response to germ-layer differentiation signalsERP155689Niels MenezesElisabetta FerrettiAdrija KalvisaNiels Alvaro Menezes, Kathryn Johanna Peterson, Xiaogang Guo, Veronica Castiglioni, Adrija Kalvisa, Katarzyna Filimonow, Karen Schachter, Luca Mariani and Elisabetta FerrettifalseWhole Genome Bisulfite Sequencing of CLDN6High vs CLDN6Low Sorted EpiSCs and APSD, and WT vs Pbx1-KO EpiSCsThe experiment was designed to assess the DNA methylation differences between CLDN6Low vs CLDN6High EpiSCs and APSD, as well as WT vs Pbx1-KO EpiSCs. Based on analysis of RNAseq data and GO, we narrowed down our focus to DNA methylation effects influencing lineage biases. We performed WGBS analysis using two different methodologies. The EZ DNA Methylation-Lightning Kit (Zymo Research, #D5031) for bisulfite conversion of CLDN6Low versus CLDN6High sorted EpiSCs and APSD cells. The next steps of library preparation was done using NEBNext Ultra II DNA Library Prep Kit (E7645S), enzymatic fragmentation using NEBNext dsDNA Fragmentase (M0348S), and sensitive adaptors NEBNext Multiplex Oligos (Methylated Adaptor, E7535S), as per manufacturer’s instructions. Samples were sequenced on NextSeq2000 (200 cycles kit), averaging 256M reads per sample. WT versus Pbx1-KO EpiSCs were treated via the post-bisulfite library construction method using Illumina’s TruSeq DNA Methylation Kit (EGMK81312) with sensitive adaptors (EGIDX81312) following the manufacturer’s instructions.2025-04-28T00:00:00Z2026-02-28T03:08:34.578Z2023-12-01T12:19:26.382ZE-MTAB-13574ERP155689EFO_0002944EFO_0004170EFO_0003789EFO_0002761EFO_0004917EFO_0005518EFO_0003816EFO_0004184EFO_0003969