Whole genome bisulfite sequencing (WGBS) from E18.5 wild-type and Tet3 knockout intestinal epithelium

Treatment protocol: The villi of E18.5 intestinal epithelium was isolated as follows. Briefly, the small intestine was dissected from E18.5 embryos, opened longitudinally, and placed on ice-cold PBS containing 30mM EDTA and 1.5mM DTT for 20min on ice. Next, the intestine was transferred to a tube containing 30mM EDTA in PBS, incubated at 37ºC for 10min and, then, vigorously shaken for 30s to release the intestinal epithelium from the lamina propia. After removing the intact intestinal muscle, the villi suspension was used for DNA extraction and WGBS. Extraction protocol Genomic DNA was extracted using QIAamp DNA micro kit (Qiagen) and bisulfite treated using EZ DNA Methylation Gold Kit (Zymo Research). Genomic DNA spiked with lambda DNA were fragmented to 200-400 bp. The DNA fragments were bisulfite treated to generate single strand DNA. Methylation sequencing adapters were ligated, followed by double strand DNA synthesis. The library was ready after size selection and PCR amplification.