title: Whole Genome Bisulfite Sequencing of CLDN6High vs CLDN6Low Sorted EpiSCs and APSD, and WT vs Pbx1-KO EpiSCs
design: Whole Genome Bisulfite Sequencing of CLDN6High vs CLDN6Low Sorted EpiSCs and APSD, and WT vs Pbx1-KO EpiSCs
library_construction_protocol: 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.  Post-sorting, cells were reseeded into Fibronectin pre-coated 6-well cell culture plates at around 10.5 x 103 cells/cm2. 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) 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.  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.