GSM8426254: E75Ect_Ctrl_WGBS_rep3; Mus musculus; Bisulfite-Seq
DNA was extracted from large amount of cells, or embryos or low input number of cells were lysed in STEM-seq lysis buffer and subject to bisulfite conversion and subsequent library preparation. For cell samples, genomic DNA was first extracted using HiPure Universal DNA Kit (Magen, D3018-02), and was then subjected to bisulfite conversion using EpiArt DNA Methylation Bisulfite Kit (Vazyme, EM101) according to the manufacturer's instructions. For embryo samples, Epi or ExE were lysed with 10 μl of STEM-seq lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1 mM EDTA, pH 8.0, 0.5% NP-40) containing 1 μl of protease K (NEB, P8107S) for 3 h at 55 °C, followed by heat-inactivation for 1 h at 72 °C. Samples were then treated with 1 μl of dsDNA Fragmentase (NEB; M0348AA) for 30 min before being subjected to bisulfite conversion using EZ DNA Methylation-Lightning Kit (ZYMO, D5030). Libraries were prepared using the EpiArt DNA Methylation Library Kit (Vazyme, NE103) according to the manufacturer's instructions. In brief, a 3' adaptor was first added to the converted and denatured ssDNA. After DNA extension and purification, a 5' adaptor was added. dsDNA with both 3' and 5' adaptors were then subjected to PCR amplification and libraries were sequenced using Illumina Hiseq X Ten or Novaseq 6000 system. For all samples, λ-DNA (Promega, D150A) was added with a mass ratio of 1:200 before bisulfite conversion for conversion rate analysis.