Whole Genome Bisulfite Sequencing of CLDN6High vs CLDN6Low Sorted EpiSCs and APSD, and WT vs Pbx1-KO EpiSCs

The 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.